Novel peptides that bind to the erythropoietin receptor

ABSTRACT

The present invention relates to peptide compounds that are agonists of the erythropoietin receptor (EPO-R). The invention also relates to therapeutic methods using such peptide compounds to treat disorders associated with insufficient or defective red blood cell production. Pharmaceutical compositions, which comprise the peptide compounds of the invention, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is claimed to U.S. provisional application Ser. Nos. 60/469,993and 60/470,244 both filed on May 12, 2003. The contents of thesepriority applications are incorporated into the present disclosure byreference and in their entireties.

FIELD OF THE INVENTION

The present invention relates to peptide compounds that are agonists ofthe erythropoietin receptor (EPO-R). The invention also relates totherapeutic methods using such peptide compounds to treat disordersassociated with insufficient or defective red blood cell production.Pharmaceutical compositions, which comprise the peptide compounds of theinvention, are also provided.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a glycoprotein hormone of 165 amino acids, witha molecular weight of about 34 kilodaltons (kD) and preferredglycosylation sites on amino-acid positions 24, 38, 83, and 126. It isinitially produced as a precursor protein with a signal peptide of 23amino acids. EPO can occur in three forms: α, β, and asialo. The α and βforms differ slightly in their carbohydrate components, but have thesame potency, biological activity, and molecular weight. The asialo formis an α or β form with the terminal carbohydrate (sialic acid) removed.The DNA sequences encoding EPO have been reported [U.S. Pat. No.4,703,008 to Lin].

EPO stimulates mitotic division and differentiation of erythrocyteprecursor cells, and thus ensures the production of erythrocytes. It isproduced in the kidney when hypoxic conditions prevail. DuringEPO-induced differentiation of erythrocyte precursor cells, globinsynthesis is induced; heme complex synthesis is stimulated; and thenumber of ferritin receptors increases. These changes allow the cell totake on more iron and synthesize functional hemoglobin, which in matureerythrocytes binds oxygen. Thus, erythrocytes and their hemoglobin playa key role in supplying the body with oxygen. These changes areinitiated by the interaction of EPO with an appropriate receptor on thecell surface of the erythrocyte precursor cells [See, e.g., Graber andKrantz (1978) Ann. Rev. Med. 29.51-66].

EPO is present in very low concentrations in plasma when the body is ina healthy state wherein tissues receive sufficient oxygenation from theexisting number of erythrocytes. This normal low concentration issufficient to stimulate replacement of red blood cells which are lostnormally through aging.

The amount of EPO in the circulation is increased under conditions ofhypoxia when oxygen transport by blood cells in the circulation isreduced. Hypoxia may be caused, for example, by substantial blood lossthrough hemorrhage, destruction of red blood cells by over-exposure toradiation, reduction in oxygen intake due to high altitude or prolongedunconsciousness, or various forms of anemia. In response to such hypoxicstress, elevated EPO levels increase red blood cell production bystimulating the proliferation of erythroid progenitor cells. When thenumber of red blood cells in circulation is greater than needed fornormal tissue oxygen requirements, EPO levels in circulation aredecreased.

Because EPO is essential in the process of red blood cell formation,this hormone has potentially useful applications in both the diagnosisand the treatment of blood disorders characterized by low or defectivered blood cell production. Recent studies have provided a basis for theprojection of EPO therapy efficacy for a variety of disease states,disorders, and states of hematologic irregularity, including:beta-thalassemia [see, Vedovato, et al. (1984) Acta. Haematol.71:211-213]; cystic fibrosis [see, Vichinsky, et al. (1984) J. Pediatric105:15-21]; pregnancy and menstrual disorders [see, Cotes, et al. (193)Brit. J. Ostet. Gyneacol. 90:304-311]; early anemia of prematurity [see,Haga, et al. (1983) Acta Pediatr. Scand. 72; 827-831]; spinal cordinjury [see, Claus-Walker, et al. (1984) Arch. Phys. Med. Rehabil.65:370-374]; space flight [see, Dunn, et al. (1984) Eur. J. Appl.Physiol. 52:178-182]; acute blood loss [see, Miller, et al. (1982) Brit.J. Haematol. 52:545-590]; aging [see, Udupa, et al. (1984) J. Lab. Clin.Med. 103:574-580 and 581-588 and Lipschitz, et al. (1983) Blood63:502-509]; various neoplastic disease states accompanied by abnormalerythropoiesis [see, Dainiak, et al. (1983) Cancer 5:1101-1106 andSchwartz, et al. (1983) Otolaryngol. 109:269-272]; and renalinsufficiency [see, Eschbach. et al. (1987) N. Eng. J. Med. 316:73-78].

Purified, homogeneous EPO has been characterized [U.S. Pat. No.4,677,195 to Hewick]. A DNA sequence encoding EPO was purified, cloned,and expressed to produce recombinant polypeptides with the samebiochemical and immunological properties and natural EPO. A recombinantEPO molecule with oligosaccharides identical to those on natural EPO hasalso been produced [See, Sasaki, et al. (1987) J. Biol. Chem.262:12059-12076].

The biological effect of EPO appears to be mediated, in part, throughinteraction with a cell membrane bound receptor. Initial studies, usingimmature erythroid cells isolated from mouse spleen, suggested that theEPO-binding cell surface proteins comprise two polypeptides havingapproximate molecular weights of 85,000 Daltons and 100,000 Daltons,respectively [Sawyer, et al. (1987) Proc. Natl. Acad. Sci. USA84:3690-3694]. The number of EPO-binding sites was calculated to averagefrom 800 to 1000 per cell surface. Of these binding sites, approximately300 bound EPO with a K_(d) of approximately 90 pM (picomolar), while theremaining bound EPO with a reduced affinity of approximately 570 pM[Sawyer, et al. (1987) J. Biol. Chem. 262:5554-5562]. An independentstudy suggested that EPO-responsive splenic erythroblasts, prepared frommice injected with the anemic strain (FVA) of the Friend leukemia virus,possess at total of approximately 400 high and a low affinity EPObinding sites with K_(d) values of approximately 100 pM and 800 pM,respectively [Landschulz, et al. (1989) Blood 73:1476-1486].

Subsequent work indicated that the two forms of EPO receptor (EPO-R)were encoded by a single gene. This gene has been cloned [See, e.g.,Jones, et al. (1990) Blood 76, 31-35; Noguchi, et al. (1991) Blood78:2548-2556; Maouche, et al. (1991) Blood 78:2557-2563]. For example,the DNA sequences and encoded peptide sequences for murine and humanEPO-R proteins are described in PCT Pub. No. WO 90/08822 to D'Andrea, etal. Current models suggest that binding of EPO to EPO-R results in thedimerization and activation of two EPO-R molecules, which results insubsequent steps of signal transduction [See, e.g., Watowich, et al.(1992) Proc. Natl. Acad. Sci. USA 89:2140-2144].

The availability of cloned genes for EPO-R facilitates the search foragonists and antagonists of this important receptor. The availability ofthe recombinant receptor protein allows the study of receptor-ligandinteraction in a variety of random and semi-random peptide diversitygeneration systems. These systems include the “peptides on plasmids”system [described in U.S. Pat. No. 6,270;170]; the “peptides on phage”system [described in U.S. Pat. No. 5,432,018 and Cwirla, et al. (1990)Proc. Natl. Acad. Sci. USA 87:6378-6382]; the “encoded syntheticlibrary” (ESL) system [described in U.S. patent application Ser. No.946,239, filed Sep. 16, 1992]; and the “very large scale immobilizedpolymer synthesis” system [described in U.S. Pat. No. 5,143,854; PCTPub. No. 90/15070; Fodor, et al. (1991) Science 251:767-773; Dower andFodor (1991) Ann. Rep. Med. Chem. 26:271-180; and U.S. Pat. No.5,424,186].

Peptides that interact to a least some exent with EPO-R have beenidentified and are described, for example in U.S. Pat. Nos. 5,773,569;5,830,851; and 5,986,047 to Wrighton, et al.; PCT Pub. No. WO 96/40749to Wrighton, et al.; U.S. Pat. No. 5,767,078 and PCT Pub. No. 96/40772to Johnson and Zivin; PCT Pub. No. WO 01/38342 to Balu; and WO 01/91780to Smith-Swintosky, et al. In particular, a group of peptides containinga peptide motif has been identified, members of which bind to EPO-R andstimulate EPO-dependent cell proliferation. Yet, peptides peptidesidentified to date that contain the motif stimulate EPO-dependent cellproliferation in vitro with EC50 values of about 20 nanomolar (nM) toabout 250 nM. Thus, peptide concentrations of 20 nM to 250 nM arerequired to stimulate 50% of the maximal cell proliferation stimulatedby EPO.

Given the immense potential of EPO-R agonists, both for studies of theimportant biological activities mediated by this receptor and fortreatment of disease, there remains a need for the identification ofpeptide EPO-R agonists of enhanced potency and activity. The presentinvention provides such compounds.

The citation and/or discussion of cited references in this section andthroughout the specification is provided merely to clarify thedescription of the present invention and is not an admission that anysuch reference is “prior art” to the present invention.

SUMMARY OF THE INVENTION

The present invention provides novel peptide compounds, which are EPO-Ragonists of dramatically enhanced potency and activity. These peptidecompounds are homodimers of peptide monomers having the amino acidsequence (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1), or homodimers ofpeptide monomers having the amino acid sequence(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2), homodimers ofpeptide monomers having the amino acid sequence(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG) (SEQ ID NO: 3); where each amino acidis indicated by standard one letter abbreviation, “(AcG)” isN-acetylglycine, “(1-nal)” is 1-naphthylalanine, and “(MeG)” isN-methylglycine, also known as sarcosine. Each peptide monomer of apeptide dimer contains an intramolecular disulfide bond between thecysteine residues of the monomer.

The peptide monomers may be dimerized by covalent attachment to abranched tertiary amide linker. The tertiary amide linker can bedepicted as:—C¹O—CH₂—X—CH₂—C²O—where: X is NCO—(CH₂)₂—N¹H—; C¹ of the linker forms an amide bond withthe ε-amino group of the C-terminal lysine residue of the first peptidemonomer; C² of the linker forms an amide bond with the ε-amino group ofthe C-terminal lysine residue of the second peptide monomer; and N¹ of Xis attached via a carbamate linkage or an amide linkage to an activatedpolyethylene glycol (PEG) moiety, where the PEG has a molecular weightof about 20,000 to about 40,000 Daltons (the term “about” indicatingthat in preparations of PEG, some molecules will weigh more, some less,than the stated molecular weight).

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N¹ of the linker isattached via a carbamate linkage to an activated polyethylene glycol(PEG) moiety, the novel peptide compounds of the invention may berepresented as follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N¹ of the linker isattached via an amide linkage to an activated polyethylene glycol (PEG)moiety, the novel peptide compounds of the invention may be representedas follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and N¹ of the linkeris attached via a carbamate linkage to an activated polyethylene glycol(PEG) moiety, the novel peptide compounds of the invention may berepresented as follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and N¹ of the linkeris attached via an amide linkage to an activated polyethylene glycol(PEG) moiety, the novel peptide compounds of the invention may berepresented as follows:

The peptide monomers may also be dimerized by covalent attachment to abranched tertiary amide linker. The tertiary amide linker can bedepicted as:—C¹O—CH₂—X—CH₂—C²O—where: X is NCO—(CH₂)₂—NH—C³O —; C¹ of the linker forms an amide bondwith the ε-amino group of the C-terminal lysine residue of the firstpeptide monomer; and C² of the linker forms an amide bond with theε-amino group of the C-terminal lysine residue of the second peptidemonomer. The peptide dimers of the invention further comprise a spacermoiety of the following structure:—N¹H—(CH₂)₄—C⁴H—N²H—where: C⁴ of the spacer is covalently bonded to C³ of X; N¹ of thespacer is covalently attached via a carbamate or an amide linkage to anactivated polyethylene glycol (PEG) moiety; and N² of the spacer iscovalently attached via a carbamate or an amide linkage to an activatedPEG moiety, where PEG has a molecular weight of about 10,000 to about50,000 Daltons (the term “about” indicating that in preparations of PEG,some molecules will weigh more, some less, than the stated molecularweight). Each PEG moiety may be, individually, 10,000 Daltons (10 kD),20 kD, 30 kD, 40 kD, or 50 kD.

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both N¹ and N² of thespacer are covalently attached via a carbamate linkage to an activatedPEG moiety, the novel peptide compounds of the invention may berepresented as follows:

In preferred embodiments, the C-terminal lysine of the two peptidemonomers is L-lysine. Also, those skilled in the art will appreciatefrom the above chemical structures that the lwo linear PEG moieities arejoined by lysine (e.g., as mPEG₂-Lys-NHS or as mPEG₂-Lysinol-NPC), whichis also preferably L-lysine and giving rise to the followingstereochemistry.

Alternatively, one or more of the lysine residues can be a D-lysine,giving rise to alternative stereochemistries which will be readilyappreciate by those skilled in the art.

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both N¹ and N² of thespacer are covalently attached via an amide linkage to an activated PEGmoiety, the novel peptide compounds of the invention may be represented,as follows:

Again, the lysine molecules in this compound are preferably allL-lysine, giving rise to the following stereochemistry.

Alternatively, one or more of the lysine residues can be a D-lysine,giving rise to alternative stereochemistries which will be readilyappreciated by those skilled in the art.

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and both N¹ and N² ofthe spacer are covalently attached via a carbamate linkage to anactivated PEG moiety, wherein Y is a carbamate group, the novel peptidecompounds of the invention may be represented as follows:

Preferably, the lysine residues joining the peptide monomer and linearPEG moieties in this molecule are all L-lysine, giving rise to thefollowing stereochemistry:

Alternatively, one or more of the lysine residues can be a D-lysine,giving rise to alternative stereochemistries that will be readilyappreciated by those of ordinary skill in the art.

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and both N¹ and N² ofthe spacer are covalently attached via an amide linkage to an activatedPEG moiety, the novel peptide compounds of the invention may berepresented as follows:

Preferably, the lysine residues joining the peptide monomer and linearPEG moieties of this molecule are all L-lysine, giving rise to thefollowing stereochemistry.

In other embodiments, one or more of the lysine residues can be aD-lysine, giving rise to alternative stereochemistries that will bereadily appreciated by persons of ordinary skill in the art.

The peptide monomers may also be dimerized by attachment to a lysinelinker, whereby one peptide monomer is attached at its C-terminus to thelysine's ε-amino group and the second peptide monomer is attached at itsC-terminus to the lysine's α-amino group.

The peptide dimers of the invention further comprise a spacer moiety ofthe following structure:—N¹H—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—N²H—At one end, N¹ of the spacer is attached via an amide linkage to acarbonyl carbon of the lysine linker. At the opposite end, N² of thespacer is attached via a carbamate linkage or an amide linkage to anactivated polyethylene glycol (PEG) moiety, where the PEG has amolecular weight of about 20,000 to about 40,000 Daltons (the term“about” indicating that in preparations of PEG, some molecules willweigh more, some less, than the stated molecular weight).

Where the spacer is attached via a carbamate linkage to an activatedpolyethylene glycol (PEG) moiety, the novel peptide compounds of theinvention may be represented as follows:

Where the spacer is attached via an amide linkage to an activatedpolyethylene glycol (PEG) moiety, the novel peptide compounds of theinvention may be represented as follows:

The invention further provides pharmaceutical compositions comprised ofsuch peptide compounds, and methods to treat various medical conditionsusing such peptide compounds.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G. The unconventional amino acids in peptidesare abbreviated as follows: 1-naphthylalanine is 1-nal or Np;N-methylglycine (also known as sarcosine) is MeG or Sc; and acetylatedglycine (N-acetylglycine) is AcG.

As used herein, the term “polypeptide” or “protein” refers to a polymerof amino acid monomers that are alpha amino acids joined togetherthrough amide bonds. Polypeptides are therefore at least two amino acidresidues in length, and are usually longer. Generally, the term“peptide” refers to a polypeptide that is only a few amino acid residuesin length. The novel EPO-R agonist peptides of the present invention arepreferably no more than about 50 amino acid residues in length. They aremore preferably of about 17 to about 40 amino acid residues in length. Apolypeptide, in contrast with a peptide, may comprise any number ofamino acid residues. Hence, the term polypeptide included peptides aswell as longer sequences of amino acids.

As used herein, the phrase “pharmaceutically acceptable” refers tomolecular entities and compositions that are “generally regarded assafe”, e.g., that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water or aqueous solution saline solutions and aqueousdextrose and glycerol solutions are preferably employed as carriers,particularly for injectable solutions. Suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

As used herein the term “agonist” refers to a biologically active ligandwhich binds to its complementary biologically active receptor andactivates the latter either to cause a biological response in thereceptor, or to enhance preexisting biological activity of the receptor.

Novel Peptides that are EPO-R Agonists

The present invention provides novel peptide compounds, which are EPO-Ragonists of dramatically enhanced potency and activity. These peptidecompounds are homodimers of peptide monomers having the amino acidsequence (AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1), or homodimers ofpeptide monomers having the amino acid sequence(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2); where each aminoacid is indicated by standard one letter abbreviation, “(AcG)” isN-acetylglycine, “(1-nal)” is 1-naphthylalanine, and “(MeG)” is “(MeG)”is N-methylglycine, also known as sarcosine. Each peptide monomer of apeptide dimer contains an intramolecular disulfide bond between thecysteine residues of the monomer. Such monomers may be representedschematically as follows:

These monomeric peptides are dimerized to provide peptide dimers ofenhanced EPO-R agonist activity. The linker (L_(K)) moiety is a branchedteriary amide, which bridges the C-termini of two peptide monomers, bysimultaneous attachment to the C-terminal lysine residue of eachmonomer. The tertiary amide linker can be depicted as:—C¹O—CH₂—X—CH₂—C²O—where: X is NCO—(CH₂)₂—N¹H—; C¹ of the linker forms an amide bond withthe ε-amino group of the C-terminal lysine residue of the first peptidemonomer; C² of the linker forms an amide bond with the ε-amino group ofthe C-terminal lysine residue of the second peptide monomer; and N¹ of Xis attached via a carbamate linkage or an amide linkage to an activatedpolyethylene glycol (PEG) moiety, where the PEG has a molecular weightof about 20,000 to about 40,000 Daltons (the term “about” indicatingthat in preparations of PEG, some molecules will weigh more, some less,than the stated molecular weight).

The tertiary amide linker may also be depicted as:—C¹O—CH₂—X—CH₂—C²O—where: X is NCO—(CH₂)₂—NH—C³—; C¹ of the linker forms an amide bond withthe ε-amino group of the C-terminal lysine residue of the first peptidemonomer; and C² of the linker forms an amide bond with the ε-amino groupof the C-terminal lysine residue of the second peptide monomer. Thepeptide dimers of the invention further comprise a spacer moiety of thefollowing structure:—N¹H—(CH₂)₄—C⁴H—N²H—where: C⁴ of the spacer is covalently bonded to C³ of X; N¹ of thespacer is covalently attached via a carbamate or an amide linkage to anactivated PEG moiety; and N² of the spacer is covalently attached via acarbamate or an amide linkage to an activated PEG moiety, where PEG hasa molecular weight of about 10,000 to about 60,000 Daltons (the term“about” indicating that in preparations of PEG, some molecules willweigh more, some less, than the stated molecular weight).

Thus, the novel peptides of the invention can also contain a PEG moiety,which is covalently attached via a carbamate linkage or an amide linkageto the tertiary amide linker of the peptide dimer. PEG is a watersoluble polymer that is pharmaceutically acceptable. PEG for use in thepresent invention may be linear, unbranched PEG having a molecularweight of about 20 kilodaltons (20K) to about 60K (the term “about”indicating that in preparations of PEG, some molecules will weigh more,some less, than the stated molecular weight). Most preferably, the PEGhas a molecular weight of about 30K to about 40K. One skilled in the artwill be able to select the desired polymer size based on suchconsiderations as the desired dosage; circulation time; resistance toproteolysis; effects, if any, on biological activity; ease in handling;degree or lack of antigenicity; and other known effects of PEG on atherapeutic peptide.

Peptides, peptide dimers and other peptide-based molecules of theinvention can be attached to water-soluble polymers (e.g., PEG) usingany of a variety of chemistries to link the water-soluble polymer(s) tothe receptor-binding portion of the molecule (e.g., peptide+spacer). Atypical embodiment employs a single attachment junction for covalentattachment of the water soluble polymer(s) to the receptor-bindingportion, however in alternative embodiments multiple attachmentjunctions may be used, including further variations wherein differentspecies of water-soluble polymer are attached to the receptor-bindingportion at distinct attachment junctions, which may include covalentattachment junction(s) to the spacer and/or to one or both peptidechains. In some embodiments, the dimer or higher order multimer willcomprise distinct species of peptide chain (i.e., a heterodimer or otherheteromultimer). By way of example and not limitation, a dimer maycomprise a first peptide chain having a PEG attachment junction and thesecond peptide chain may either lack a PEG attachment junction orutilize a different linkage chemistry than the first peptide chain andin some variations the spacer may contain or lack a PEG attachmentjunction and said spacer, if PEGylated, may utilize a linkage chemistrydifferent than that of the first and/or second peptide chains. Analternative embodiment employs a PEG attached to the spacer portion ofthe receptor-binding portion and a different water-soluble polymer(e.g., a carbohydrate) conjugated to a side chain of one of the aminoacids of the peptide portion of the molecule.

A wide variety of polyethylene glycol (PEG) species may be used forPEGylation of the receptor-binding portion (peptides+spacer).Substantially any suitable reactive PEG reagent can be used. Inpreferred embodiments, the reactive PEG reagent will result in formationof a carbamate or amide bond upon conjugation to the receptor-bindingportion. Suitable reactive PEG species include, but are not limited to,those which are available for sale in the Drug Delivery Systems catalog(2003) of NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu4-chome, Shibuya-ku, Tokyo 150-6019) and the Molecular Engineeringcatalog (2003) of Nektar Therapeutics (490 Discovery Drive, Huntsville,Ala. 35806). For example and not limitation, the following PEG reagentsare often preferred in various embodiments: mPEG2-NHS, mPEG2-ALD,multi-Arm PEG, mPEG(MAL)₂, mPEG2(MAL), mPEG-NH2, mPEG-SPA, mPEG-SBA,mPEG-thioesters, mPEG-Double Esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET,heterofunctional PEGs (NH2-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS,NHS-PEG-VS, NHS-PEG-MAL), PEG acrylates (ACRL-PEG-NHS),PEG-phospholipids (e.g., mPEG-DSPE), multiarmed PEGs of the SUNBRITEseries including the GL series of glycerine-based PEGs activated by achemistry chosen by those skilled in the art, any of the SUNBRITEactivated PEGs (including but not limited to carboxyl-PEGs, p-NP-PEGs,Tresyl-PEGs, aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs,maleimido-PEGs, hydroxyl-PEG-amine, amino-PEG-COOH,hydroxyl-PEG-aldehyde, carboxylic anhydride type-PEG, functionalizedPEG-phospholipid, and other similar and/or suitable reactive PEGs asselected by those skilled in the art for their particular applicationand usage.

The novel peptides of the invention can also contain two PEG moietiesthat are covalently attached via a carbamate or an amide linkage to aspacer moiety, wherein the spacer moiety is covalently bonded to thetertiary amide linker of the peptide dimer. Each of the two PEG moietiesused in such embodiments of the present invention may be linear and maybe linked together at a single point of attachment. Each PEG moietypreferably has a molecular weight of about 10 kilodaltons (10K) to about60K (the term “about” indicating that in preparations of PEG, somemolecules will weigh more, some less, than the stated molecular weight).Linear PEG moieties are particularly preferred. More preferably, each ofthe two PEG moieties has a molecular weight of about 20K to about 40K,and still more preferably between about 20K and about 40K. Still morepreferably, each of the two PEG moieties has a molecular weight of about20K. One skilled in the art will be able to select the desired polymersize based on such considerations as the desired dosage; circulationtime; resistance to proteolysis; effects, if any, on biologicalactivity; ease in handling; degree or lack of antigenicity; and otherknown effects of PEG on a therapeutic peptide.

The present invention also comprises peptide agonists that arehomodimers of peptide monomers having the amino acid sequence(AcG)GLYACHMGPIF(1-nal)VCQPLR(MeG) (SEQ ID NO: 3), where each amino acidis indicated by standard one letter abbreviation, “(AcG)” isN-acetylglycine, “(1-nal)” is 1-naphthylalanine, and “(MeG)” isN-methylglycine, also known as sarcosine. Each peptide monomer of thepeptide dimer contains an intramolecular disulfide bond between thecysteine residues of the monomer. Such monomers may be representedschematically as follows:

These monomeric peptides are dimerized to provide peptide dimers ofenhanced EPO-R agonist activity. The linker (L_(K)) moiety is a lysineresidue, which bridges the C-termini of two peptide monomers, bysimultaneous attachment to the C-terminal amino acid of each monomer.One peptide monomer is attached at its C-terminus to the lysine'sε-amino group and the second peptide monomer is attached at itsC-terminus to the lysine's α-amino group. For example, the dimer may beillustrated structurally as shown in Formula I, and summarized as shownin Formula II:

In Formula I and Formula II, N² represents the nitrogen atom of lysine'sε-amino group and N¹ represents the nitrogen atom of lysine's α-aminogroup.

The peptide dimers of the invention further comprise a spacer moiety ofthe following structure:—N¹H—(CH₂)₂—O—(CH₂)₂—O—(CH₂)₂—N²H—At one end, N¹ of the spacer is attached via an amide linkage to acarbonyl carbon of the lysine linker. At the opposite end, N² of thespacer is attached via a carbamate linkage or an amide linkage to anactivated polyethylene glycol (PEG) moiety, where the PEG has amolecular weight of about 10,000 to about 60,000 Daltons (the term“about” indicating that in preparations of PEG, some molecules willweigh more, some less, than the stated molecular weight). Morepreferably, the PEG has a molecular weight of about 20,000 to 40,000Daltons.

Thus, the novel peptides of the invention also contain a PEG moiety,which is covalently attached to the peptide dimer. PEG is a watersoluble polymer that is pharmaceutically acceptable. PEG for use in thepresent invention may be linear, unbranched PEG having a molecularweight of about 20 kilodaltons (20K) to about 60K (the term “about”indicating that in preparations of PEG, some molecules will weigh more,some less, than the stated molecular weight). Most preferably, the PEGhas a molecular weight of about 20K to about 40K, and still morepreferably a molecular weight of about 30K to about 40K. One skilled inthe art will be able to select the desired polymer size based on suchconsiderations as the desired dosage; circulation time; resistance toproteolysis; effects, if any, on biological activity; ease in handling;degree or lack of antigenicity; and other known effects of PEG on atherapeutic peptide.

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N¹ of the linker isattached via a carbamate linkage to an activated polyethylene glycol(PEG) moiety, the novel peptide compounds of the invention may berepresented as follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and N¹ of the linker isattached via an amide linkage to an activated polyethylene glycol (PEG)moiety, the novel peptide compounds of the invention may be representedas follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and N¹ of the linkeris attached via a carbamate linkage to an activated polyethylene glycol(PEG) moiety, the novel peptide compounds of the invention may berepresented as follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and N¹ of the linkeris attached via an amide linkage to an activated polyethylene glycol(PEG) moiety, the novel peptide compounds of the invention may berepresented as follows:

Preferred peptide dimers of the present invention include, but are notlimited to:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both N¹ and N² of thespacer are covalently attached via a carbamate linkage to an activatedPEG moiety, the novel peptide compounds of the invention may berepresented as follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLRK (SEQ ID NO: 1) and both N¹ and N² of thespacer are covalently attached via an amide linkage to an activated PEGmoiety, the novel peptide compounds of the invention may be representedas follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and both N¹ and N² ofthe spacer are covalently attached via a carbamate linkage to anactivated PEG moiety, the novel peptide compounds of the invention maybe represented as follows:

Where each monomer of the homodimer has the amino acid sequence,(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) and both N¹ and N² ofthe spacer are covalently attached via an amide linkage to an activatedPEG moiety, the novel peptide compounds of the invention may berepresented as follows:

Preferred peptide dimers of the present invention include, but are notlimited to:

Where the spacer is attached via a carbamate linkage to an activatedpolyethylene glycol (PEG) moiety, the novel peptide compounds of theinvention may be represented as follows:

Where the spacer is attached via an amide linkage to an activatedpolyethylene glycol (PEG) moiety, the novel peptide compounds of theinvention may be represented as follows:

This dimeric structure can be written [Ac-peptide,disulfide]₂Lys-spacer-PEG_(20-40K) to denote an N-terminally acetylatedpeptide bound to both the α and ε amino groups of lysine with eachpeptide containing an intramolecular disulfide loop and a spacermolecule forming a covalent linkage between the C-terminus of lysine anda PEG moiety, where the PEG has a molecular weight of about 20,000 toabout 40,000 Daltons.

Preferred peptide dimers of the present invention include, but are notlimited to:

Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as a,a-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for compounds of the present invention.Examples of unconventional amino acids include, but are not limited to:β-alanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine,N-methylglycine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, norleucine, and other similar amino acids and iminoacids. Other modifications are also possible, including modification ofthe amino terminus, modification of the carboxy terminus, replacement ofone or more of the naturally occurring genetically encoded amino acidswith an unconventional amino acid, modification of the side chain of oneor more amino acid residues, peptide phosporylation, and the like.

The peptide sequences of the present invention and be present alone orin conjunction with N-terminal and/or C-terminal extensions of thepeptide chain. Such extensions may be naturally encoded peptidesequences optionally with or substantially without non-naturallyoccurring sequences; the extensions may include any additions,deletions, point mutations, or other sequence modifications orcombinations as desired by those skilled in the art. For example and notlimitation, naturally-occurring sequences may be full-length or partiallength and may include amino acid substitutions to provide a site forattachment of carbohydrate, PEG, other polymer, or the like via sidechain conjugation. In a variation, the amino acid substitution resultsin humanization of a sequence to make in compatible with the humanimmune system. Fusion proteins of all types are provided, includingimmunoglobulin sequences adjacent to or in near proximity to the EPO-Ractivating sequences of the present invention with or without anon-immunoglobulin spacer sequence. One type of embodiment is animmunoglobulin chain having the EPO-R activating sequence in place ofthe variable (V) region of the heavy and/or light chain.

Preparation of the Peptide Compounds of the Invention:

Peptide Synthesis

The peptides of the invention may be prepared by classical methods knownin the art. These standard methods include exclusive solid phasesynthesis, partial solid phase synthesis methods, fragment condensation,classical solution synthesis, and recombinant DNA technology [See, e.g.,Merrifield J. Am. Chem. Soc. 1963 85:2149].

In one embodiment, the peptide monomers of a peptide dimer aresynthesized individually and dimerized subsequent to synthesis.

In another embodiment, the peptide monomers of a dimer are linked viatheir C-termini by a branched tertiary amide linker L_(K) moiety havingtwo functional groups capable of serving as initiation sites for peptidesynthesis and a third functional group (e.g., a carboxyl group or anamino group) that enables binding to another molecular moiety (e.g., asmay be present on the surface of a solid support). In this case, the twopeptide monomers may be synthesized directly onto two reactive nitrogengroups of the linker L_(K) moiety in a variation of the solid phasesynthesis technique. Such synthesis may be sequential or simultaneous.

In another embodiment, the two peptide monomers may be synthesizeddirectly onto two reactive nitrogen groups of the linker L_(K) moiety ina variation of the solid phase synthesis technique. Such synthesis maybe sequential or simultaneous. In this embodiment, a lysine linker(L_(K)) moiety having two amino groups capable of serving as initiationsites for peptide synthesis and a third functional group (e.g., thecarboxyl group of a lysine; or the amino group of a lysine amide, alysine residue wherein the carboxyl group has been converted to an amidemoiety —CONH₂) that enables binding to another molecular moiety (e.g.,as may be present on the surface of a solid support) is used.

Where sequential synthesis of the peptide chains of a dimer onto alinker is to be performed, two amine functional groups on the linkermolecule are protected with two different orthogonally removable amineprotecting groups. The protected linker is coupled to a solid supportvia the linker's third functional group. The first amine protectinggroup is removed, and the first peptide of the dimer is synthesized onthe first deprotected amine moiety. Then the second amine protectinggroup is removed, and the second peptide of the dimer is synthesized onthe second deprotected amine moiety. For example, the first amino moietyof the linker may be protected with Alloc, and the second with Fmoc. Inthis case, the Fmoc group (but not the Alloc group) may be removed bytreatment with a mild base [e.g., 20% piperidine in dimethyl formamide(DMF)], and the first peptide chain synthesized. Thereafter the Allocgroup may be removed with a suitable reagent [e.g., Pd(PPh₃)/4-methylmorpholine and chloroform], and the second peptide chain synthesized.Note that where different thiol-protecting groups for cysteine are to beused to control disulfide bond formation (as discussed below) thistechnique must be used even where the final amino acid sequences of thepeptide chains of a dimer are identical.

Where simultaneous synthesis of the peptide chains of a dimer onto alinker is to be performed, two amine functional groups of the linkermolecule are protected with the same removable amine protecting group.The protected linker is coupled to a solid support via the linker'sthird functional group. In this case the two protected functional groupsof the linker molecule are simultaneously deprotected, and the twopeptide chains simultaneously synthesized on the deprotected amines.Note that using this technique, the sequences of the peptide chains ofthe dimer will be identical, and the thiol-protecting groups for thecysteine residues are all the same.

A preferred method for peptide synthesis is solid phase synthesis. Solidphase peptide synthesis procedures are well-known in the art [see, e.g.,Stewart Solid Phase Peptide Syntheses (Freeman and Co.: San Francisco)1969; 2002/2003 General Catalog from Novabiochem Corp, San Diego, USA;Goodman Synthesis of Peptides and Peptidomimetics (Houben-Weyl,Stuttgart) 2002]. In solid phase synthesis, synthesis is typicallycommenced from the C-terminal end of the peptide using an α-aminoprotected resin. A suitable starting material can be prepared, forinstance, by attaching the required α-amino acid to a chloromethylatedresin, a hydroxymethyl resin, a polystyrene resin, a benzhydrylamineresin, or the like. One such chloromethylated resin is sold under thetrade name BIO-BEADS SX-1 by Bio Rad Laboratories (Richmond, Calif.).The preparation of the hydroxymethyl resin has been described[Bodonszky, et al. (1966) Chem. Ind. London 38:1597]. Thebenzhydrylamine (BHA) resin has been described [Pietta and Marshall(1970) Chem. Commun. 650], and the hydrochloride form is commerciallyavailable from Beckman Instruments, Inc. (Palo Alto, Calif.). Forexample, an α-amino protected amino acid may be coupled to achloromethylated resin with the aid of a cesium bicarbonate catalyst,according to the method described by Gisin (1973) Helv. Chim. Acta56:1467.

After initial coupling, the α-amino protecting group is removed, forexample, using trifluoroacetic acid (TFA) or hydrochloric acid (HCl)solutions in organic solvents at room temperature. Thereafter, α-aminoprotected amino acids are successively coupled to a growingsupport-bound peptide chain. The α-amino protecting groups are thoseknown to be useful in the art of stepwise synthesis of peptides,including: acyl-type protecting groups (e.g., formyl, trifluoroacetyl,acetyl), aromatic urethane-type protecting groups [e.g.,benzyloxycarboyl (Cbz) and substituted Cbz], aliphatic urethaneprotecting groups [e.g., t-butyloxycarbonyl (Boc), isopropyloxycarbonyl,cyclohexyloxycarbonyl], and alkyl type protecting groups (e.g., benzyl,triphenylmethyl), fluorenylmethyl oxycarbonyl (Fmoc), allyloxycarbonyl(Alloc), and 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde).

The side chain protecting groups (typically ethers, esters, trityl, PMC,and the like) remain intact during coupling and is not split off duringthe deprotection of the amino-terminus protecting group or duringcoupling. The side chain protecting group must be removable upon thecompletion of the synthesis of the final peptide and under reactionconditions that will not alter the target peptide. The side chainprotecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl,benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. The side chain protectinggroups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl, andcyclohexyl. The side chain protecting groups for Thr and Ser includeacetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl,and Cbz. The side chain protecting groups for Arg include nitro, Tosyl(Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts),2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf),4-mthoxy-2,3,6-trimethyl-benzenesulfonyl (Mtr), or Boc. The side chainprotecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl(2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-Br-Cbz), Tos, or Boc.

After removal of the α-amino protecting group, the remaining protectedamino acids are coupled stepwise in the desired order. Each protectedamino acid is generally reacted in about a 3-fold excess using anappropriate carboxyl group activator such as2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate(HBTU) or dicyclohexylcarbodimide (DCC) in solution, for example, inmethylene chloride (CH₂Cl₂), N-methylpyrrolidone, dimethyl formamide(DMF), or mixtures thereof.

After the desired amino acid sequence has been completed, the desiredpeptide is decoupled from the resin support by treatment with a reagent,such as trifluoroacetic acid (TFA) or hydrogen fluoride (HF), which notonly cleaves the peptide from the resin, but also cleaves all remainingside chain protecting groups. When a chloromethylated resin is used,hydrogen fluoride treatment results in the formation of the free peptideacids. When the benzhydrylamine resin is used, hydrogen fluoridetreatment results directly in the free peptide amide. Alternatively,when the chloromethylated resin is employed, the side chain protectedpeptide can be decoupled by treatment of the peptide resin with ammoniato give the desired side chain protected amide or with an alkylamine togive a side chain protected alkylamide or dialkylamide. Side chainprotection is then removed in the usual fashion by treatment withhydrogen fluoride to give the free amides, alkylamides, ordialkylamides.

In preparing the esters of the invention, the resins used to prepare thepeptide acids are employed, and the side chain protected peptide iscleaved with base and the appropriate alcohol (e.g., methanol). Sidechain protecting groups are then removed in the usual fashion bytreatment with hydrogen fluoride to obtain the desired ester.

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of any of thecompounds of the invention. Synthetic amino acids that can besubstituted into the peptides of the present invention include, but arenot limited to, N-methyl, L-hydroxypropyl, L-3, 4-dihydroxyphenylalanyl,δ amino acids such as L-δ-hydroxylysyl and D-δ-methylalanyl,L-α-methylalanyl, β amino acids, and isoquinolyl. D-amino acids andnon-naturally occurring synthetic amino acids can also be incorporatedinto the peptides of the present invention.

Peptide Modifications

One can also modify the amino and/or carboxy termini of the peptidecompounds of the invention to produce other compounds of the invention.For example, the amino terminus may be acetylated with acetic acid or ahalogenated derivative thereof such as α-chloroacetic acid,α-bromoacetic acid, or α-iodoacetic acid).

One can replace the naturally occurring side chains of the 20genetically encoded amino acids (or the stereoisomeric D amino acids)with other side chains, for instance with groups such as alkyl, loweralkyl, cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl,amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and the lowerester derivatives thereof, and with 4-, 5-, 6-, to 7-memberedheterocyclic. In particular, proline analogues in which the ring size ofthe proline residue is changed from 5 members to 4, 6, or 7 members canbe employed. Cyclic groups can be saturated or unsaturated, and ifunsaturated, can be aromatic or non-aromatic. Heterocyclic groupspreferably contain one or more nitrogen, oxygen, and/or sulfurheteroatoms. Examples of such groups include the furazanyl, furyl,imidazolidinyl, imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl,morpholinyl (e.g. morpholino), oxazolyl, piperazinyl (e.g.,1-piperazinyl), piperidyl (e.g., 1-piperidyl, piperidino), pyranyl,pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,pyrimidinyl, pyrrolidinyl (e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl,thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.,thiomorpholino), and triazolyl. These heterocyclic groups can besubstituted or unsubstituted. Where a group is substituted, thesubstituent can be alkyl, alkoxy, halogen, oxygen, or substituted orunsubstituted phenyl.

One can also readily modify peptides by phosphorylation, and othermethods [e.g., as described in Hruby, et al. (1990) Biochem J.268:249-262].

The peptide compounds of the invention also serve as structural modelsfor non-peptidic compounds with similar biological activity. Those ofskill in the art recognize that a variety of techniques are availablefor constructing compounds with the same or similar desired biologicalactivity as the lead peptide compound, but with more favorable activitythan the lead with respect to solubility, stability, and susceptibilityto hydrolysis and proteolysis [See, Morgan and Gainor (1989) Ann. Rep.Med. Chem. 24:243-252]. These techniques include replacing the peptidebackbone with a backbone composed of phosphonates, amidates, carbamates,sulfonamides, secondary amines, and N-methylamino acids.

Formation of Disulfide Bonds

The compounds of the present invention contain two intramoleculardisulfide bonds. Such disulfide bonds may be formed by oxidation of thecysteine residues of each peptide monomer.

In one embodiment, the control of cysteine bond formation is exercisedby choosing an oxidizing agent of the type and concentration effectiveto optimize formation of the desired isomer. For example, oxidation of apeptide dimer to form two intramolecular disulfide bonds (one on eachpeptide chain) is preferentially achieved (over formation ofintermolecular disulfide bonds) when the oxidizing agent is DMSO oriodine (12).

In other embodiments, the formation of cysteine bonds is controlled bythe selective use of thiol-protecting groups during peptide synthesis.For example, where a dimer with two intramolecular disulfide bonds isdesired, the first monomer peptide chain is synthesized with the twocysteine residues of the core sequence protected with a first thiolprotecting group [e.g., trityl(Trt), allyloxycarbonyl (Alloc), and1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde)or the like],then the second monomer peptide is synthesized the two cysteine residuesof the core sequence protected with a second thiol protecting groupdifferent from the first thiol protecting group [e.g., acetamidomethyl(Acm), t-butyl (tBu), or the like]. Thereafter, the first thiolprotecting groups are removed effecting bisulfide cyclization of thefirst monomer, and then the second thiol protecting groups are removedeffecting bisulfide cyclization of the second monomer.

Other embodiments of this invention provide for analogues of thesedisulfide derivatives in which one of the sulfurs has been replaced by aCH₂ group or other isotere for sulfur. These analogues can be preparedfrom the compounds of the present invention, wherein each peptidemonomer contains at least one C or homocysteine residue and anα-amino-γ-butyric acid in place of the second C residue, via anintramolecular or intermolecular displacement, using methods known inthe art [See, e.g., Barker, et al. (1992) J. Med. Chem. 35:2040-2048 andOr, et al. (1991) J. Org. Chem. 56:3146-3149]. One of skill in the artwill readily appreciate that this displacement can also occur usingother homologs of α-amino-γ-butyric acid and homocysteine.

In addition to the foregoing cyclization strategies, other non-disulfidepeptide cyclization strategies can be employed. Such alternativecyclization strategies include, for example, amide-cyclizationstrategies as well as those involving the formation of thio-ether bonds.Thus, the compounds of the present invention can exist in a cyclizedform with either an intramolecular amide bond or an intramolecularthio-ether bond. For example, a peptide may be synthesized wherein onecysteine of the core sequence is replaced with lysine and the secondcysteine is replaced with glutamic acid. Thereafter a cyclic monomer maybe formed through an amide bond between the side chains of these tworesidues. Alternatively, a peptide may be synthesized wherein onecysteine of the core sequence is replaced with lysine (or serine). Acyclic monomer may then be formed through a thio-ether linkage betweenthe side chains of the lysine (or serine) residue and the secondcysteine residue of the core sequence. As such, in addition to disulfidecyclization strategies, amide-cyclization strategies and thio-ethercyclization strategies can both be readily used to cyclize the compoundsof the present invention. Alternatively, the amino-terminus of thepeptide can be capped with an α-substituted acetic acid, wherein theα-substituent is a leaving group, such as an α-haloacetic acid, forexample, α-chloroacetic acid, α-bromoacetic acid, or α-iodoacetic acid.

Addition of Branched Tertiary Amide Linker

The peptide monomers may be dimerized by a branched tertiary amidelinker moeity. In one embodiment, the linker is incorporated into thepeptide during peptide synthesis. For example, where a linker L_(K)moiety contains two functional groups capable of serving as initiationsites for peptide synthesis and one or more other functional groups(e.g., a carboxyl group or an amino group) that enables binding to oneor more other molecular moieties, the linker may be conjugated to asolid support. Thereafter, two peptide monomers may be synthesizeddirectly onto the two reactive nitrogen groups of the linker L_(K)moiety in a variation of the solid phase synthesis technique.

In alternate embodiments, the linker may be conjugated to the twopeptide monomers of a peptide dimer after peptide synthesis. Suchconjugation may be achieved by methods well established in the art. Inone embodiment, the linker contains two functional groups suitable forattachment to the target functional groups of the synthesized peptidemonomers. For example, a linker containing two carboxyl groups, eitherpreactivated or in the presence of a suitable coupling reagent, may bereacted with the target lysine side chain amine groups of each of twopeptide monomers.

For example, the peptide momomers may be chemically coupled to thetertiary amide linker,A*-C¹O—CH₂—X—CH₂—C²O—B*where: X is NCO—(CH₂)₂—NH—Y and Y is a suitable protecting group, suchas a t-butyloxycarbonyl (Boc) protecting group; A* is a suitablefunctional group, such as N-oxy succinimide, used to conjugate C¹ of thelinker to the 8-amino group of the C-terminal lysine residue of thefirst peptide monomer; and B* is a suitable functional group, such asN-oxy succinimide, used to conjugate C² of the linker to the 8-aminogroup of the C-terminal lysine residue of the second peptide monomer.

Additionally, for example, the peptide monomers may be chemicallycoupled to the tertiary amide linker,A*-C¹O—CH₂—X—CH₂—C²O—B*where: X is NCO—(CH₂)₂—NH—C³O—; A* is a suitable functional group, suchas N-oxy succinimide, used to conjugate C¹ of the linker to the 8-aminogroup of the C-terminal lysine residue of the first peptide monomer; andB* is a suitable functional group, such as N-oxy succinimide, used toconjugate C² of the linker to the ε-amino group of the C-terminal lysineresidue of the second peptide monomer; and the tertiary amide linker ischemically bonded to the spacer moiety,Y—NH—(CH₂)₄—C⁴H—NH—Ywhere: C³ of X is covalently bonded to C⁴ of the spacer; and Y is asuitable protecting group, such as a t-butyloxycarbonyl (Boc) protectinggroup.Addition of Lysine Linker

The peptide monomers may be dimerized by a lysine linker L_(K) moiety.In one embodiment, the lysine linker in incorporated into the peptideduring peptide synthesis. For example, where a lysine linker L_(K)moiety contains two functional groups capable of serving as initiationsites for peptide synthesis and a third functional group (e.g., acarboxyl group or an amino group) that enables binding to anothermolecular moiety, the linker may be conjugated to a solid support.Thereafter, two peptide monomers may be synthesized directly onto thetwo reactive nitrogen groups of the lysine linker L_(K) moiety in avariation of the solid phase synthesis technique.

In alternate embodiments where a peptide dimer is dimerized by a lysinelinker L_(K) moiety, said linker may be conjugated to the two peptidemonomers of a peptide dimer after peptide synthesis. Such conjugationmay be achieved by methods well established in the art. In oneembodiment, the linker contains at least two functional groups suitablefor attachment to the target functional groups of the synthesizedpeptide monomers. For example, a the lysine's two free amine groups maybe reacted with the C-terminal carboxyl groups of each of two peptidemonomers.

Addition of Spacer

The peptide compounds of the invention further comprise a spacer moiety.In one embodiment the spacer may be incorporated into the peptide duringpeptide synthesis. For example, where a spacer contains a free aminogroup and a second functional group (e.g., a carboxyl group or an aminogroup) that enables binding to another molecular moiety, the spacer maybe conjugated to the solid support.

In one embodiment, a spacer containing two functional groups is firstcoupled to the solid support via a first functional group. Next thelysine linker L_(K) moiety having two functional groups capable ofserving as initiation sites for peptide synthesis and a third functionalgroup (e.g., a carboxyl group or an amino group) that enables binding toanother molecular moiety is conjugated to the spacer via the spacer'ssecond functional group and the linker's third functional group.Thereafter, two peptide monomers may be synthesized directly onto thetwo reactive nitrogen groups of the linker L_(K) moiety in a variationof the solid phase synthesis technique. For example, a solid supportcoupled spacer with a free amine group may be reacted with a lysinelinker via the linker's free carboxyl group.

In alternate embodiments the spacer may be conjugated to the peptidedimer after peptide synthesis. Such conjugation may be achieved bymethods well established in the art. In one embodiment, the linkercontains at least one functional group suitable for attachment to thetarget functional group of the synthesized peptide. For example, aspacer with a free amine group may be reacted with a peptide'sC-terminal carboxyl group. In another example, a linker with a freecarboxyl group may be reacted with the free amine group of a lysineamide.

Attachment of Polyethylene Glycol (PEG)

In recent years, water-soluble polymers, such as polyethylene glycol(PEG), have been used for the covalent modification of peptides oftherapeutic and diagnostic importance. Attachment of such polymers isthought to enhance biological activity, prolong blood circulation time,reduce immunogenicity, increase aqueous solubility, and enhanceresistance to protease digestion. For example, covalent attachment ofPEG to therapeutic polypeptides such as interleukins [Knauf, et al.(1988) J. Biol. Chem. 263;15064; Tsutsumi, et al. (1995) J. ControlledRelease 33:447), interferons (Kita, et al. (1990) Drug Des. Delivery6:157), catalase (Abuchowski, et al. (1977) J. Biol. Chem. 252:582),superoxide dismutase (Beauchamp, et al. (1983) Anal. Biochem. 131:25),and adenosine deaminase (Chen, et al. (1981) Biochim. Biophy. Acta660:293), has been reported to extend their half life in vivo, and/orreduce their immunogenicity and antigenicity.

The peptide compounds of the invention may comprise a polyethyleneglycol (PEG) moiety, which is covalently attached to the branchedtertiary amide linker or the spacer of the peptide dimer via a carbamatelinkage or via an amide linkage. An example of PEG used in the presentinvention is linear, unbranched PEG having a molecular weight of about20 kiloDaltons (20K) to about 40K (the term “about” indicating that inpreparations of PEG, some molecules will weigh more, some less, than thestated molecular weight). Preferably, the PEG has a molecular weight ofabout 30K to about 40K.

Another example of PEG used in the present invention is linear PEGhaving a molecular weight of about 10K to about 60K (the term “about”indicating that in preparations of PEG, some molecules will weigh more,some less, than the stated molecular weight). Preferably, the PEG has amolecular weight of about 20K to about 40K. More preferably, the PEG hasa molecular weight of about 20K.

Examples of methods for covalent attachment of PEG (PEGylation) aredescribed below. These illustrative descriptions are not intended to belimiting. One of ordinary skill in the art will appreciate that avariety of methods for covalent attachment of a broad range of PEG iswell established in the art. As such, peptide compounds to which PEG hasbeen attached by any of a number of attachment methods known in the artare encompassed by the present invention.

For example, PEG may be covalently bound to the linker via a reactivegroup to which an activated PEG molecule may be bound (e.g., a freeamino group or carboxyl group). PEG molecules may be attached to aminogroups using methoxylated PEG (“mPEG”) having different reactivemoieties. Such polymers include mPEG-succinimidyl succinate,mPEG-succinimidyl carbonate, mPEG-imidate, mPEG-4-nitrophenyl carbonate,and mPEG-cyanuric chloride. Similarly, PEG molecules may be attached tocarboxyl groups using methoxylated PEG with a free amine group(mPEG-NH₂).

In some embodiments, the linker or spacer contains a terminal aminogroup (i.e., positioned at the terminus of the spacer). This terminalamino group may be reacted with a suitably activated PEG molecule, suchas mPEG-para-nitrophenylcarbonate (mPEG-NPC), to make a stable covalentcarbamate bond. Alternatively, this terminal amino group may be reactedwith a suitably activated PEG molecule, such as an mPEG-succinimidylbutyrate (mPEG-SBA) or mPEG-succinimidyl propionate (mPEG-SPA)containing a reactive N-hydroxl-succinimide (NHS) group, to make astable covalent carbamate bond. In other embodiments, the linkerreactive group contains a carboxyl group capable of being activated toform a covalent bond with an amine-containing PEG molecule undersuitable reaction conditions. Suitable PEG molecules include mPEG-NH₂and suitable reaction conditions include carbodiimide-mediated amideformation or the like.

EPO-R Agonist Activity Assays:

In Vitro Functional Assays

In vitro competitive binding assays quantitate the ability of a testpeptide to compete with EPO for binding to EPO-R. For example (see,e.g., as described in U.S. Pat. No. 5,773,569), the extracellular domainof the human EPO-R (EPO binding protein, EBP) may be recombinantlyproduced in E. coli and the recombinant protein coupled to a solidsupport, such as a microtitre dish or a synthetic bead [e.g., Sulfolinkbeads from Pierce Chemical Co. (Rockford, EL)]. Immobilized EBP is thenincubated with labeled recombinant EPO, or with labeled recombinant EPOand a test peptide. Serial dilutions of test peptide are employed forsuch experiments. Assay points with no added test peptide define totalEPO binding to EBP. For reactions containing test peptide, the amount ofbound EPO is quantitated and expressed as a percentage of the control(total=100%) binding. These values are plotted versus peptideconcentration. The IC50 value is defined as the concentration of testpeptide which reduces the binding of EPO to EBP by 50% (i.e., 50%inhibition of EPO binding).

A different in vitro competitive binding assay measures the light signalgenerated as a function of the proximity of two beads: an EPO-conjugatedbead and an EPO-R-conjugated bead. Bead proximity is generated by thebinding of EPO to EPO-R. A test peptide that competes with EPO forbinding to EPO-R will prevent this binding, causing a decrease in lightemission. The concentration of test peptide that results in a 50%decrease in light emission is defined as the IC50 value.

The peptides of the present invention compete very efficiently with EPOfor binding to the EPO-R. This enhanced function is represented by theirability to inhibit the binding of EPO at substantially lowerconcentrations of peptide (i.e., they have very low IC50 values).

The biological activity and potency of monomeric and dimeric peptideEPO-R agonists of the invention, which bind specifically to theEPO-receptor, may be measured using in vitro cell-based functionalassays.

One assay is based upon a murine pre-B-cell line expressing human EPO-Rand further transfected with a fos promoter-driven luciferase reportergene construct. Upon exposure to EPO or another EPO-R agonist, suchcells respond by synthesizing luciferase. Luciferase causes the emissionof light upon addition of its substrate luciferin. Thus, the level ofEPO-R activation in such cells may be quantitated via measurement ofluciferase activity. The activity of a test peptide is measured byadding serial dilutions of the test peptide to the cells, which are thenincubated for 4 hours. After incubation, luciferin substrate is added tothe cells, and light emission is measured. The concentration of testpeptide that results in a half-maximal emission of light is recorded asthe EC50.

The peptides of the present invention show dramatically enhanced abilityto promote EPO-R signaling-dependent luciferase expression in thisassay. This enhanced function is represented by their ability to yieldhalf of the maximal luciferase activity at substantially lowerconcentrations of peptide (i.e., they have very low EC50 values). Thisassay is a preferred method for estimating the potency and activity ofan EPO-R agonist peptide of the invention.

Another assay may be performed using FDC-P1/ER cells [Dexter, et al.(1980) J. Exp. Med. 152:1036-1047], a well characterized nontransformedmurine bone marrow derived cell line into which EPO-R has been stablytransfected. These cells exhibit EPO-dependent proliferation.

In one such assay, the cells are grown to half stationary density in thepresence of the necessary growth factors (see, e.g., as described inU.S. Pat. No. 5,773,569). The cells are then washed in PBS and starvedfor 16-24 hours in whole media without the growth factors. Afterdetermining the viability of the cells (e.g., by trypan blue staining),stock solutions (in whole media without the growth factors) are made togive about 10⁵ cells per 50 μL. Serial dilutions of the peptide EPO-Ragonist compounds (typically the free, solution phase peptide as opposedto a phage-bound or other bound or immobilized peptide) to be tested aremade in 96-well tissue culture plates for a final volume of 50 μL perwell. Cells (50 μL) are added to each well and the cells are incubated2448 hours, at which point the negative controls should die or bequiescent. Cell proliferation is then measured by techniques known inthe art, such as an MTT assay which measures H³-thymidine incorporationas an indication of cell proliferation [see, Mosmann (1983) J. Immunol.Methods 65:55-63]. Peptides are evaluated on both the EPO-R-expressingcell line and a parental non-expressing cell line. The concentration oftest peptide necessary to yield one half of the maximal cellproliferation is recorded as the EC50.

The peptides of the present invention show dramatically enhanced abilityto promote EPO-dependent cell growth in this assay. This enhancedfunction is represented by their ability to yield half of the maximalcell proliferation stimulation activity at substantially lowerconcentrations of peptide (i.e., they have very low EC50 values). Thisassay is a preferred method for estimating the potency and activity ofan EPO-R agonist peptide of the invention.

In another assay, the cells are grown to stationary phase inEPO-supplemented medium, collected, and then cultured for an additional18 hr in medium without EPO. The cells are divided into three groups ofequal cell density: one group with no added factor (negative control), agroup with EPO (positive control), and an experimental group with thetest peptide. The cultured cells are then collected at various timepoints, fixed, and stained with a DNA-binding fluorescent dye (e.g.,propidium iodide or Hoechst dye, both available from Sigma).Fluorescence is then measured, for example, using a FACS Scan Flowcytometer. The percentage of cells in each phase of the cell cycle maythen be determined, for example, using the SOBR model of CelIFITsoftware (Becton Dickinson). Cells treated with EPO or an active peptidewill show a greater proportion of cells in S phase (as determined byincreased fluorescence as an indicator of increased DNA content)relative to the negative control group.

Similar assays may be performed using FDCP-1 [see, e.g., Dexter et al.(1980) J. Exp. Med. 152:1036-1047] or TF-1 [Kitamura, et al. (1989)Blood 73:375-380] cell lines. FDCP-1 is a growth factor dependent murinemulti-potential primitive hematopoietic progenitor cell line that canproliferate, but not differentiate, when supplemented withWEHI-3-conditioned media (a medium that contains IL-3, ATCC numberTIB-68). For such experiments, the FDCP-1 cell line is transfected withthe human or murine EPO-R to produce FDCP-1-hEPO-R or FDCP-1-mEPO-R celllines, respectively, that can proliferate, but not differentiate, in thepresence of EPO. TF-1, an EPO-dependent cell line, may also be used tomeasure the effects of peptide EPO-R agonists on cellular proliferation.

In yet another assay, the procedure set forth in Krystal (1983) Exp.Hematol 11:649-660 for a microassay based on H³-thymidine incorporationinto spleen cells may be employed to ascertain the ability of thecompounds of the present invention to serve as EPO agonists. In brief,B6C3F₁ mice are injected daily for two days with phenylhydrazine (60mg/kg). On the third day, spleen cells are removed and their ability toproliferate over a 24 hour period ascertained using an MTT assay.

The binding of EPO to EPO-R in an erythropoietin-responsive cell lineinduces tyrosine phosphorylation of both the receptor and numerousintracellular proteins, including Shc, vav and JAK2 kinase. Therefore,another in vitro assay measures the ability of peptides of the inventionto induce tyrosine phosphorylation of EPO-R and downstream intracellularsignal transducer proteins. Active peptides, as identified by bindingand proliferation assays described above, elicit a phosphorylationpattern nearly identical to that of EPO in erythropoietin-responsivecells. For this assay, FDC-P1/ER cells [Dexter, et al. (1980) J Exp Med152:1036-47] are maintained in EPO-supplemented medium and grown tostationary phase. These cells are then cultured in medium without EPOfor 24 hr. A defined number of such cells is then incubated with a testpeptide for approximately 10 min at 37° C. A control sample of cellswith EPO is also run with each assay. The treated cells are thencollected by centrifugation, resuspended in SDS lysis buffer, andsubjected to SDS polyacrylamide gel electrophoresis. The electrophoresedproteins in the gel are transferred to nitrocellulose, and thephosphotyrosine containing proteins on the blot visualized by standardimmunological techniques. For example, the blot may be probed with ananti-phosphotyrosine antibody (e.g., mouse anti-phosphotyrosine IgG fromUpstate Biotechnology, Inc.), washed, and then probed with a secondaryantibody [e.g., peroxidase labeled goat anti-mouse IgG from Kirkegaard &Perry Laboratories, Inc. (Washington, D.C.)]. Thereafter,phosphotyrosine-containing proteins may be visualized by standardtechniques including colorimetric, chemiluminescent, or fluorescentassays. For example, a chemiluminescent assay may be performed using theECL Western Blotting System from Amersham.

Another cell-based in vitro assay that may be used to assess theactivity of the peptides of the present invention is a colony assay,using murine bone marrow or human peripheral blood cells. Murine bonemarrow may be obtained from the femurs of mice, while a sample of humanperipheral blood may obtained from a healthy donor. In the case ofperipheral blood, mononuclear cells are first isolated from the blood,for example, by centrifugation through a Ficoll-Hypaque gradient [StemCell Technologies, Inc. (Vancouver, Canada)]. For this assay a nucleatedcell count is performed to establish the number and concentration ofnucleated cells in the original sample. A defined number of cells isplated on methyl cellulose as per manufacturer's instructions [Stem CellTechnologies, Inc. (Vancouver, Canada)]. An experimental group istreated with a test peptide, a positive control group is treated withEPO, and a negative control group receives no treatment. The number ofgrowing colonies for each group is then scored after defined periods ofincubation, generally 10 days and 18 days. An active peptide willpromote colony formation.

Other in vitro biological assays that can be used to demonstrate theactivity of the compounds of the present invention are disclosed inGreenberger, et al. (1983) Proc. Natl. Acad. Sci. USA 80:2931-2935(EPO-dependent hematopoietic progenitor cell line); Quelle andWojchowski (1991) J. Biol. Chem. 266:609-614 (protein tyrosinephosphorylation in B6SUt.EP cells); Dusanter-Fourt, et al. (1992) J.Biol. Chem. 287:10670-10678 (tyrosine phosphorylation of EPO-receptor inhuman EPO-responsive cells); Quelle, et al. (1992) J. Biol. Chem.267:17055-17060 (tyrosine phosphorylation of a cytosolic protein, pp100, in FDC-ER cells); Worthington, et al. (1987) Exp. Hematol. 15:85-92(colorimetric assay for hemoglobin); Kaiho and Miuno (1985) Anal.Biochem. 149:117-120 (detection of hemoglobin with 2,7-diaminofluorene);Patel, et al. (1992) J. Biol. Chem. 267:21300-21302 (expression ofc-myb); Witthuhn, et al. (1993) Cell 74:227-236 (association andtyrosine phosphorylation of JAK2); Leonard, et al. (1993) Blood82:1071-1079 (expression of GATA transcription factors); and Ando, etal. (1993) Proc. Natl. Acad. Sci. USA 90:9571-9575 (regulation of GItransition by cycling D2 and D3).

An instrument designed by Molecular Devices Corp., known as amicrophysiometer, has been reported to be successfully used formeasurement of the effect of agonists and antagonists on variousreceptors. The basis for this apparatus is the measurement of thealterations in the acidification rate of the extracellular media inresponse to receptor activation.

In Vivo Functional Assays

One in vivo functional assay that may be used to assess the potency of atest peptide is the polycythemic exhypoxic mouse bioassay. For thisassay, mice are subjected to an alternating conditioning cycle forseveral days. In this cycle, the mice alternate between periods ofhypobaric conditions and ambient pressure conditions. Thereafter, themice are maintained at ambient pressure for 2-3 days prior toadministration of test samples. Test peptide samples, or EPO standard inthe case positive control mice, are injected subcutaneously into theconditioned mice. Radiolabeled iron (e.g., Fe⁵⁹) is administered 2 dayslater, and blood samples taken two days after administration ofradiolabeled iron. Hematocrits and radioactivity measurements are thendetermined for each blood sample by standard techniques. Blood samplesfrom mice injected with active test peptides will show greaterradioactivity (due to binding of Fe⁵⁹ by erythrocyte hemoglobin) thanmice that did not receive test peptides or EPO.

Another in vivo functional assay that may be used to assess the potencyof a test peptide is the reticulocyte assay. For this assay, normaluntreated mice are subcutaneously injected on three consecutive dayswith either EPO or test peptide. On the third day, the mice are alsointraperitoneally injected with iron dextran. At day five, blood samplesare collected from the mice. The percent (%) of reticulocytes in theblood is determined by thiazole orange staining and flow cytometeranalysis (retic-count program). In addition, hematocrits are manuallydetermined. The percent of corrected reticulocytes is determined usingthe following formula:% RETIC _(CORRECTED)=% RETIC _(OBSERVED)X(Hematocrit_(INDIVIDUAL)/Hematocrit_(NORMAL))Active test compounds will show an increased % RETIC_(CORRECTED) levelrelative to mice that did not receive test peptides or EPO.Use of EPO-R Agonist Peptides of the Invention

The peptide compounds of the invention are useful in vitro as tools forunderstanding the biological role of EPO, including the evaluation ofthe many factors thought to influence, and be influenced by, theproduction of EPO and the binding of EPO to the EPO-R (e.g., themechanism of EPO/EPO-R signal transduction/receptor activation). Thepresent peptides are also useful in the development of other compoundsthat bind to the EPO-R, because the present compounds provide importantstructure-activity-relationship information that facilitate thatdevelopment.

Moreover, based on their ability to bind to EPO-R, the peptides of thepresent invention can be used as reagents for detecting EPO-R on livingcells; fixed cells; in biological fluids; in tissue homogenates; inpurified, natural biological materials; etc. For example, by labelingsuch peptides, one can identify cells having EPO-R on their surfaces. Inaddition, based on their ability to bind EPO-R, the peptides of thepresent invention can be used in in situ staining, FACS(fluorescence-activated cell sorting) analysis, Western blotting, ELISA(enzymelinked immunosorbent assay), etc. In addition, based on theirability to bind to EPO-R, the peptides of the present invention can beused in receptor purification, or in purifying cells expressing EPO-R onthe cell surface (or inside permeabilized cells).

The peptides of the invention can also be utilized as commercialreagents for various medical research and diagnostic purposes. Such usescan include but are not limited to: (1) use as a calibration standardfor quantitating the activities of candidate EPO-R agonists in a varietyof functional assays; (2) use as blocking reagents in random peptidescreening, i.e., in looking for new families of EPO-R peptide ligands,the peptides can be used to block recovery of EPO peptides of thepresent invention; (3) use in co-crystallization with EPO-R, i.e.,crystals of the peptides of the present invention bound to the EPO-R maybe formed, enabling determination of receptor/peptide structure by X-raycrystallography; (4) use to measure the capacity of erythrocyteprecursor cells induce globin synthesis and heme complex synthesis, andto increase the number of ferritin receptors, by initiatingdifferentiation; (5) use to maintain the proliferation and growth ofEPO-dependent cell lines, such as the FDCP-1-mEPO-R and the TF-1 celllines; (6) use related to labeling the peptides of the invention with aradioactive chromophore; and (7) other research and diagnosticapplications wherein the EPO-R is preferably activated or suchactivation is conveniently calibrated against a known quantity of anEPO-R agonist, and the like.

In yet another aspect of the present invention, methods of treatment andmanufacture of a medicament are provided. The peptide compounds of theinvention may be administered to warm blooded animals, including humans,to simulate the binding of EPO to the EPO-R in vivo. Thus, the presentinvention encompasses methods for therapeutic treatment of disordersassociated with a deficiency of EPO, which methods compriseadministering a peptide of the invention in amounts sufficient tostimulate the EPO-R and thus, alleviate the symptoms associated with adeficiency of EPO in vivo. For example, the peptides of this inventionwill find use in the treatment of renal insufficiency and/or end-stagerenal failure/dialysis; anemia associated with AIDS; anemia associatedwith chronic inflammatory diseases (for example, rheumatoid arthritisand chronic bowel inflammation) and autoimmune disease; and for boostingthe red blood count of a patient prior to surgery. Other disease states,disorders, and states of hematologic irregularity that may be treated byadministration of the peptides of this invention include:beta-thalassemia; cystic fibrosis; pregnancy and menstrual disorders;early anemia of prematurity; spinal cord injury; space flight; acuteblood loss; aging; stroke, ischemia (both CNS and cardiac); and variousneoplastic disease states accompanied by abnormal erythropoiesis.

In other embodiments, the peptide compounds of the invention may be usedfor the treatment of disorders which are not characterized by low ordeficient red blood cells, for example as a pretreatment prior totransfusions. In addition, administration of the compounds of thisinvention can result in a decrease in bleeding time and thus, will finduse in the administration to patients prior to surgery or forindications wherein bleeding is expected to occur. In addition, thecompounds of this invention will find use in the activation ofmegakaryoctes.

Since EPO has been shown to have a mitogenic and chemotactic effect onvascular endothelial cells as well as an effect on central cholinergicneurons [see, e.g., Amagnostou, et al. (1990) Proc. Natl. Acad. Sci. USA87:5978-5982 and Konishi, et al. (1993) Brain Res. 609:29-35], thecompounds of this invention will also find use for the treatment of avariety of vascular disorders, such as: promoting wound healing;promoting growth of collateral coronary blood vessels (such as thosethat may occur after myocardial infarction); trauma treatment; andpost-vascular graft treatment. The compounds of this invention will alsofind use for the treatment of a variety of neurological disorders,generally characterized by low absolute levels of acetyl choline or lowrelative levels of acetyl choline as compared to other neuroactivesubstances e.g., neurotransmitters.

Pharmaceutical Compositions

In yet another aspect of the present invention, pharmaceuticalcompositions of the above EPO-R agonist peptide compounds are provided.Conditions alleviated or modulated by the administration of suchcompositions include those indicated above. Such pharmaceuticalcompositions may be for administration by oral, parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), transmucosal (nasal, vaginal, rectal, or sublingual)routes of administration or using bioerodible inserts and can beformulated in dosage forms appropriate for each route of administration.In general, comprehended by the invention are pharmaceuticalcompositions comprising effective amounts of an EPO-R agonist peptide,or derivative products, of the invention together with pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength;additives such as detergents and solubilizing agents (e.g., Tween 20,Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol); incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hylauronicacid may also be used. Such compositions may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the present proteins and derivatives. See, e.g., Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712 which are herein incorporated by reference.The compositions may be prepared in liquid form, or may be in driedpowder (e.g., lyophilized) form.

Oral Delivery

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets, pellets, powders, orgranules. Also, liposomal or proteinoid encapsulation may be used toformulate the present compositions (as, for example, proteinoidmicrospheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given by Marshall, K.In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes Chapter10, 1979, herein incorporated by reference. In general, the formulationwill include the EPO-R agonist peptides (or chemically modified formsthereof) and inert ingredients which allow for protection against thestomach environment, and release of the biologically active material inthe intestine.

Also contemplated for use herein are liquid dosage forms for oraladministration, including pharmaceutically acceptable emulsions,solutions, suspensions, and syrups, which may contain other componentsincluding inert diluents; adjuvants such as wetting agents, emulsifyingand suspending agents; and sweetening, flavoring, and perfuming agents.

The peptides may be chemically modified so that oral delivery of thederivative is efficacious. Generally, the chemical modificationcontemplated is the attachment of at least one moiety to the componentmolecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecomponent or components and increase in circulation time in the body. Asdiscussed above, PEGylation is a preferred chemical modification forpharmaceutical usage. Other moieties that may be used include: propyleneglycol, copolymers of ethylene glycol and propylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane[see, e.g., Abuchowski and Davis (1981) “Soluble Polymer-EnzymeAdducts,” in Enzymes as Drugs. Hocenberg and Roberts, eds.(Wiley-Interscience: New York, N.Y.) pp. 367-383; and Newmark, et al.(1982) J. Appl. Biochem. 4:185-189].

For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. One skilled in the art has available formulations which willnot dissolve in the stomach, yet will release the material in theduodenum or elsewhere in the intestine. Preferably, the release willavoid the deleterious effects of the stomach environment, either byprotection of the peptide (or derivative) or by release of the peptide(or derivative) beyond the stomach environment, such as in theintestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic (i.e. powder), for liquid forms a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The peptide (or derivative) can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs, or even as tablets.These therapeutics could be prepared by compression.

Colorants and/or flavoring agents may also be included. For example, thepeptide (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the peptide (or derivative)with an inert material. These diluents could include carbohydrates,especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may be also beused as fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. The disintegrants may also be insoluble cationicexchange resins. Powdered gums may be used as disintegrants and asbinders. and can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the peptide (or derivative) agent togetherto form a hard tablet and include materials from natural products suchas acacia, tragacanth, starch and gelatin. Others include methylcellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC)could both be used in alcoholic solutions to granulate the peptide (orderivative).

An antifrictional agent may be included in the formulation of thepeptide (or derivative) to prevent sticking during the formulationprocess. Lubricants may be used as a layer between the peptide (orderivative) and the die wall, and these can include but are not limitedto; stearic acid including its magnesium and calcium salts,polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils andwaxes. Soluble lubricants may also be used such as sodium laurylsulfate, magnesium lauryl sulfate, polyethylene glycol of variousmolecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the peptide (or derivative) into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrosefatty acid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the peptide (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Controlled release oral formulations may be desirable. The peptide (orderivative) could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation.Some enteric coatings also have a delayed release effect. Another formof a controlled release is by a method based on the Oros therapeuticsystem (Alza Corp.), i.e. the drug is enclosed in a semipermeablemembrane which allows water to enter and push drug out through a singlesmall opening due to osmotic effects.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The peptide (orderivative) could also be given in a film coated tablet and thematerials used in this instance are divided into 2 groups. The first arethe nonenteric materials and include methyl cellulose, ethyl cellulose,hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropylcellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methylcellulose, providone and the polyethylene glycols. The second groupconsists of the enteric materials that are commonly esters of phthalicacid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

Parenteral Delivery

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured using sterile water, or some other sterile injectablemedium, immediately before use.

Rectal or Vaginal Delivery

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax. Compositions fornasal or sublingual administration are also prepared with standardexcipients well known in the art.

Pulmonary Delivery

Also contemplated herein is pulmonary delivery of the EPO-R agonistpeptides (or derivatives thereof). The peptide (or derivative) isdelivered to the lungs of a mammal while inhaling and traverses acrossthe lung epithelial lining to the blood stream [see, e.g., Adjei, et al.(1990) Pharmaceutical Research 7:565-569; Adjei, et al. (1990) Int. J.Pharmaceutics 63:135-144 (leuprolide acetate); Braquet, et al. (1989) J.Cardiovascular Pharmacology 13(sup5):143-146 (endothelin-1); Hubbard, etal. (1989) Annals of Internal Medicine, Vol. III, pp. 206-212(α1-antitrypsin); Smith, et al. (1989) J. Clin. Invest. 84:1145-1146(α-1-proteinase); Oswein, et al. (1990) “Aerosolization of Proteins”,Proceedings of Symposium on Respiratory Drug Delivery II Keystone,Colorado (recombinant human growth hormone); Debs, et al. (1988) J.Immunol. 140:3482-3488 (interferon-γ and tumor necrosis factor α); andU.S. Pat. No. 5,284,656 to Platz, et al. (granulocyte colony stimulatingfactor). A method and composition for pulmonary delivery of drugs forsystemic effect is described in U.S. Pat. No. 5,451,569 to Wong, et al.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acorn II nebulizer(Marquest Medical Products, Englewood, Colo.); the Ventolin metered doseinhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhalerpowder inhaler (Fisons Corp., Bedford, Mass.).

All such devices require the use of formulations suitable for thedispensing of peptide (or derivative). Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to the usual diluents,adjuvants and/or carriers useful in therapy. Also, the use of liposomes,microcapsules or microspheres, inclusion complexes, or other types ofcarriers is contemplated. Chemically modified peptides may also beprepared in different formulations depending on the type of chemicalmodification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise peptide (or derivative) dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of thepeptide (or derivative) caused by atomization of the solution in formingthe aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the peptide (or derivative)suspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing peptide (or derivative) and mayalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. The peptide (orderivative) should most advantageously be prepared in particulate formwith an average particle size of less than 10 mm (or microns), mostpreferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal Delivery

Nasal delivery of the EPO-R agonist peptides (or derivatives) is alsocontemplated. Nasal delivery allows the passage of the peptide to theblood stream directly after administering the therapeutic product to thenose, without the necessity for deposition of the product in the lung.Formulations for nasal delivery include those with dextran orcyclodextran.

Dosages

For all of the peptide compounds, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age, and generalhealth of the recipient, will be able to ascertain proper dosing. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally dosage levels of 0.001 to 10 mg/kg of body weight daily areadministered to mammals. Generally, for intravenous injection orinfusion, dosage may be lower. The dosing schedule may vary, dependingon the circulation half-life and the formulation used.

The peptides of the present invention (or their derivatives) may beadministered in conjunction with one or more additional activeingredients or pharmaceutical compositions.

EXAMPLES

The present invention is next described by means of the followingexamples. However, the use of these and other examples anywhere in thespecification is illustrative only, and in no way limits the scope andmeaning of the invention or of any exemplified form. Likewise, theinvention is not limited to any particular preferred embodimentsdescribed herein. Indeed, many modifications and variations of theinvention may be apparent to those skilled in the art upon reading thisspecification, and can be made without departing from its spirit andscope. The invention is therefore to be limited only by the terms of theappended claims, along with the full scope of equivalents to which theclaims are entitled.

Example 1 Synthesis of EPO-R Agonist Peptide Dimers by Solid PhaseSynthesis

Step 1—Synthesis of Cbz-TAP: A solution containing the commerciallyavailable diamine (“TAP” from Aldrich Chemical Co.) (10 g, 67.47 mmol)in anhydrous DCM (100 ml) was cooled to 0° C.

A solution of benzyl chloroformate (4.82 ml, 33.7 mmol) in anhydrous DCM(50 ml) was added slowly through a dropping funnel over a period of 6-7h, maintaining the temperature of the reaction mixture at 0° C.throughout, then allowed to warm to room temperature (25° C.). After afurther 16 h, the DCM was removed under vacuum and the residuepartitioned between 3N HCl and ether. The aqueous layers were collectedand neutralized with 50% aq. NaOH to pH 8-9 and extracted with ethylacetate. The ethyl acetate layer was dried over anhydrous Na₂SO₄, thenconcentrated under vacuum to provide the crude mono-Cbz-TAP (5 g, about50% yield). This compound was used for the next reaction without anyfurther purification.

Step 2—Synthesis of Cbz-TAP-Boc: To a vigorously stirred suspension ofthe Cbz-TAP (5 g, 17.7 mmol) in hexane (25 ml) was added Boc₂O (3.86 g,17.7 mmol) and stirring continued at RT overnight. The reaction mixturewas diluted with DCM (25 ml) and washed with 10% aq. citric acid (2×),water (2×) and brine. The organic layer was dried over anhydrous Na₂SO₄and concentrated under vacuum. The crude product (yield 5 g) was useddirectly in the next reaction.

Step 3—Synthesis of Boc-TAP: The crude product from the previousreaction was dissolved in methanol (25 ml) and hydrogenated in presenceof 5% Pd on Carbon (5% w/W) under balloon pressure for 16 hrs. Themixture was filtered, washed with methanol and the filtrate concentratedin vacuo to provide the crude H-TAP-Boc product (yield 3.7 g). Theoverall approximate yield of Boc-TAP after Steps 1-3 was 44% (calculatedbased on the amount of Cbz-Cl used.)

Step 4—Synthesis of TentaGel-Linker: TentaGel bromide (2.5 g, 0.48mmol/g, from Rapp Polymere, Germany), phenolic linker (5 equivalent, andK₂CO₃ (5 equivalent) were heated in 20 mL of DMF to 70° C. for 14 h.After cooling to room temperature, the resin was washed (0.1 N HCl,water, ACN, DMF, MeOH) and dried to give an amber-colored resin.

Step 5—Synthesis of TentaGel-linker-TAP(Boc): 2.5 gms of the resin fromabove and H-TAP-Boc (1.5 gms, 5 eq.) and glacial AcOH (34 μl, 5 eq.) wastaken in a mixture of 1:1 MeOH-THF and shaken overnight. A 1M solutionof sodium cyanoborohydride (5 eq) in THF was added to this and shakenfor another 7 hrs. The resin was filtered washed (DMF, THF, 0.1 N HCl,water, MeOH) and dried. A small amount of the resin was benzoylated withBz-Cl and DIEA in DCM and cleaved with 70% TFA-DCM and checked by LCMSand HPLC.

Step 6—Synthesis of TentaGel-linker-TAP-Lys: The resin from above wastreated with a activated solution of Fmoc-Lys(Fmoc)-OH (prepared from 5eq. of amino acid and 5 eq. of HATU dissolved at 0.5 M in DMF, followedby the addition of 10 eq. of DIEA) and allowed to gently shake 14 h. Theresin was washed (DMF, THF, DCM, MeOH) and dried to yield the protectedresin. Residual amine groups were capped by treating the resin with asolution of 10% acetic anhydride, 20% pyridine in DCM for 20 minutes,followed by washing as above. The Fmoc groups are removed by gentlyshaking the resin in 30% piperideine in DMF for 20 minutes, followed bywashing (DMF, THF, DCM, MeOH) and drying.

Step 7—Synthesis of TentaGel-Linker-TAP-Lys(Peptide)₂: The resin fromabove was subjected to repeated cycles of Fmoc-amino acid couplings withHBTU/HOBt activation and Fmoc removal with piperidine to build bothpeptide chains simultaneously. This was convenently carried out on a ABI433 automated peptide synthesizer available from Applied Biosystems,Inc. After the final Fmoc removal, the terminal amine groups wereacylated with acetic anhydride (10 eq.) and DIEA (20 eq.) in DMF for 20minutes, follwed by washing as above.

Step 8—Cleavage from resin: The resin from above was suspended in asolution of TFA (82.5%), phenol (5%), ethanedithiol (2.5%), water (5%),and thioanisole (5%) for 3 h at room temperature. Alternative cleavagecocktails such as TFA (95%), water (2.5%), and triisopropylsilane (2.5%)can also be used. The TFA solution was cooled to 5° C. and poured intoEt₂O to percipatate the peptide. Filtration and drying under reducedpressure gave the desired peptide. Purification via preparative HPLCwith a C18 column afforded the pure peptide.

Step 9—Oxidation of peptides to form intramolecular disulfide bonds: Thepeptide dimer was dissolved in 20% DMSO/water (1 mg dry weightpeptide/mL) and allowed to stand at room temperature for 36 h. Thepeptide was purified by loading the reaction mixture onto a C18 HPLCcolumn (Waters Delta-Pak C18, 15 micron particle size, 300 angstrom poresize, 40 mm×200 mm length), followed by a linear ACN/water/0.01% TFAgradiant from 5 to 95% ACN over 40 minutes. Lyopholization of thefractions containing the desired peptide affords the product as a fluffywhite solid.

Step 10—PEGylation of the Terminal —NH₂ Group:PEGylation via a carbamate bond: The peptide dimer was mixed with 1.5eq. (mole basis) of activated PEG species (mPEG-NPC from NOF Corp.Japan) in dry DMF to afford a clear solution. After 5 minutes 4 eq ofDIEA was added to above solution. The mixture was stirred at ambienttemperature 14 h, followed by purification with C18 reverse phase HPLC.The structure of PEGylated peptide was confirmed by MALDI mass. Thepurified peptide was also subjected to purification via cation ionexchange chromatography as outlined below.

PEGylation via an amide bond: The peptide dimer is mixed with 1.5 eq.(mole basis) of 1 eq. activated PEG species (PEG-SPA-NHS from ShearwaterCorp, USA) in dry DMF to afford a clear solution. After 5 minutes 10 eqof DIEA is added to above solution. The mixture is stirred at ambienttemperature 2 h, followed by purification with C18 reverse phase HPLC.The structure of PEGylated peptide was confirmed by MALDI mass. Thepurified peptide was also subjected to purification via cation ionexchange chromatography as outlined below.

Step 11—Ion exchange purification: Several exchange supports weresurveyed for their ability to separate the above peptide-PEG conjugatefrom unreacted (or hydrolyzed) PEG, in addition to their ability toretain the starting dimeric peptides. The ion exchange resin (2-3 g) wasloaded into a 1 cm column, followed by conversion to the sodium form(0.2 N NaOH loaded onto column until elutant was pH 14, ca. 5 columnvolumes), and than to the hydrogen form (eluted with either 0.1 N HCl or0.1 M HOAc until elutant matched load pH, ca. 5 column volumes),followed by washing with 25% ACN/water until pH 6. Either the peptideprior to conjugation or the peptide-PEG conjugate was dissolved in 25%ACN/water (10 mg/mL) and the pH adjusted to <3 with TFA, then loaded onthe column. After washing with 2-3 column volumes of 25% ACN/water andcollecting 5 mL fractions, the peptide was released from the column byelution with 0.1 M NH₄OAc in 25% ACN/water, again collecting 5 mLfractions. Analysis via HPLC revealed which fractions contained thedesired peptide. Analysis with an Evaporative Light-Scattering Detector(ELSD) indicated that when the peptide was retained on the column andwas eluted with the NH₄OAc solution (generally between fractions 4 and10), no non-conjugated PEG was observed as a contaminant. When thepeptide eluted in the initial wash buffer (generally the first 2fractions), no separation of desired PEG-conjugate and excess PEG wasobserved.

The following columns successfully retained both the peptide and thepeptide-PEG conjugate, and successfully purified the peptide-PEGconjugate from the unconjugates peptide: TABLE 1 Ion Exhange ResinsSupport Source Mono S HR 5/5 strong cation Amersham Biosciences exchangepre-loaded column SE53 Cellulose, microgranular Whatman strong cationexchange support SP Sepharose Fast Flow strong Amersham Biosciencescation exchange support

Example 2 Synthesis of EPO-R Agonist Peptide Dimers by FragmentCondensation

Step 1—Synthesis of (Cbz)₂Lys: Lysine is reacted under standardconditions with a solution of benzyl chloroformate to obtain lysineprotected at its two amino groups with a Cbz group.

Step 2—Synthesis of Boc-TAP: Boc-TAP is syntheisized as described inSteps 1 through 3 of Example 1.

Step 3—Coupling of (Cbz)₂Lys and Boc-TAP: (Cbz)₂-Lys and Boc-TAP arecoupled under standard coupling conditions to obtain (Cbz)₂-Lys-TAP-Boc.

Step 4—Lys-TAP-Boc: The crude product from the previous reaction isdissolved in methanol (25 ml) and hydrogenated in presence of 5% Pd onCarbon (5% w/W) under balloon pressure for 16 hrs. The mixture isfiltered, washed with methanol and the filtrate concentrated in vacuo toprovide the crude Lys-TAP-Boc product

Step 5—Synthesis of peptide monomers by fragment condensation: Fourpeptide fragments of the peptide monomer sequence are synthesized bystandard techniques. These partially protected fragments are thensubjected to two independent rounds of coupling. In the first round, theN-terminal half of the monomer is formed by coupling two of the peptidefragments, while the C-terminal half of the monomer is formed bycoupling the other two of the peptide fragments. In the second round ofcoupling, the N-terminal and C-terminal halves are coupled to form thefully protected monomer. The monomer is then OBn-deprotected by standardtechniques.

Step 6—Oxidation of peptide monomers to form intramolecular disulfidebonds: The OBn-deprotected condensed peptide monomers are then oxidizedwith under iodide to form intramolecular disulfide bonds between theAcm-protected cysteine residues of the monomers.

Step 7—Coupling of Lys-TAP-Boc to oxidized OBn-deprotected monomers toform a peptide dimer: Lys-TAP-Boc is coupled to a two-fold molar excessof the oxidized OBn-deprotected monomers under standard conditions toform a peptide dimer. The peptide dimer is then deprotected understandard conditions.

-   -   Step 8—PEGylation of deprotected dimer: The deprotected peptide        dimer is then PEGylated as described in Step 10 of Example 1.    -   Step 9—Ion exchange purification: The PEGylated peptide dimer is        then purified as described in Step 11 of Example 1.

Example 3 In Vitro Activity Assays

This example describes various in vitro assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. The results for these assays demonstrate that the novelpeptides of this invention bind to EPO-R and activate EPO-R signaling.Moreover, the results for these assays show that the novel peptidecompositions exhibit a surprising increase in EPO-R binding affinity andbiological activity compared to EPO mimetic peptides that have beenpreviously described.

EPO-R agonist peptide dimers are prepared according to the methodsprovided in Example 1 or Example 2. The potency of these peptide dimersis evaluated using a series of in vitro activity assays, including: areporter assay, a proliferation assay, a competitive binding assay, anda C/BFU-e assay. These four assays are described in further detailbelow.

The results of these in vitro activity assays are summarized in Table 2.

1. Reporter Assay

This assay is based upon a on a murine pre-B-cell line derived reportercell, Baf3/EpoR/GCSFR fos/lux. This reporter cell line expresses achimeric receptor comprising the extra-cellular portion of the human EPOreceptor to the intra-cellular portion of the human GCSF receptor. Thiscell line is further transfected with a fos promoter-driven luciferasereporter gene construct. Activation of this chimeric receptor throughaddition of erythropoietic agent results in the expression of theluciferase reporter gene, and therefore the production of light uponaddition of the luciferase substrate luciferin. Thus, the level of EPO-Ractivation in such cells may be quantitated via measurement ofluciferase activity.

The Baf3/EpoR/GCSFR fos/ux cells are cultured in DMEM/F12 medium (Gibco)supplemented with 10% fetal bovine serum (FBS; Hyclone), 10% WEHI-3supernatant (the supernatant from a culture of WEHI-3 cells, ATCC #TIB-68), and penicillin/streptomycin. Approximately 18 h before theassay, cells are starved by transferring them to DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day ofassay, cells are washed once with DMEM/F12 medium supplemented with 10%FBS (no WEHI-3 supernatant), then 1×10⁶ cells/mL are cultured in thepresence of a known concentration of test peptide, or with EPO(R & DSystems Inc., Minneapolis, Minn.) as a positive control, in DMEM/F12medium supplemented with 10% FBS (no WEHI-3 supernatant). Serialdilutions of the test peptide are concurrently tested in this assay.Assay plates are incubated for 4 h at 37° C. in a 5% CO₂ atmosphere,after which luciferin (Steady-Glo; Promega, Madison, Wis.) is added toeach well. Following a 5-minute incubation, light emission is measuredon a Packard Topcount Luminometer (Packard Instrument Co., DownersGrove, Ill.). Light counts are plotted relative to test peptideconcentration and analysed using Graph Pad software. The concentrationof test peptide that results in a half-maximal emission of light isrecorded as the EC50 [See Table 2: Reporter EC50].

2. Proliferation Assay

This assay is based upon a murine pre-B-cell line, BaB, transfected toexpress human EPO-R. Proliferation of the resulting cell line,BaF3/Gal4/Elk/EPOR, is dependent on EPO-R activation. The degree of cellproliferation is quantitated using MTT, where the signal in the MTTassay is proportional to the number of viable cells.

The BaF3/Gal4/Elk/EPOR cells are cultured in spinner flasks in DMEM/F12medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3supernatant (ATCC # TIB-68). Cultured cells are starved overnight, in aspinner flask at a cell density of 1×10⁶ cells/ml, in DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cellsare then washed twice with Dulbecco's PBS (Gibco), and resuspended to adensity of 1×10⁶ cells/ml in DMEM/F12 supplemented with 10% FBS (nowEHI-3 supernatant). 50 μL aliquots (50,000 cells) of the cellsuspension are then plated, in triplicate, in 96 well assay plates. 50μL aliquots of dilution series of test EPO mimetic peptides, or 50 μLEPO(R & D Systems Inc., Minneapolis, Minn.) or Aranesp™(darbepoeitinalpha, an ERO-R agonist commerically available from Amgen) in DMEM/F12media supplemented with 10% FBS (no WEHI-3 supernatant 1) are added tothe 96 well assay plates (final well volume of 100 μL). For example, 12different dilutions may be tested where the final concentration of testpeptide (or control EPO peptide) ranges from 810 pM to 0.0045 pM. Theplated cells are then incubated for 48 h at 37° C. Next, 10 μL of MTT(Roche Diagnostics) is added to each culture dish well, and then allowedto incubate for 4 h. The reaction is then stopped by adding 10%SDS+0.01N HCl. The plates are then incubated overnight at 37° C.Absorbance of each well at a wavelenght of 595 nm is then measured byspectrophotometry. Plots of the absorbance readings versus test peptideconcentration are constructed and the EC50 calculated using Graph Padsoftware. The concentration of test peptide that results in ahalf-maximal absorbance is recorded as the EC50 [See Table 2:Proliferation EC50].

3. Competitive Binding Assay

Competitive binding calculations are made using an assay in which alight signal is generated as a function of the proximity of two beads: astreptavidin donor bead bearing a biotinylated EPO-R-binding peptidetracer and an acceptor bead to which is bound EPO-R. Light is generatedby non-radiative energy transfer, during which a singlet oxygen isreleased from a first bead upon illumination, and contact with thereleased singlet oxygen causes the second bead to emit light. These beadsets are commercially available (Packard). Bead proximity is generatedby the binding of the EPO-R-binding peptide tracer to the EPO-R. A testpeptide that competes with the EPO-R-binding peptide tracer for bindingto EPO-R will prevent this binding, causing a decrease in lightemission.

In more detail the method is as follows: Add 4 μL of serial dilutions ofthe test EPO-R agonist peptide, or positive or negative controls, towells of a 384 well plate. Thereafter, add 2 μL/well of receptor/beadcocktail. Receptor bead cocktail consists of: 15 μL of 5 mg/mlstreptavidin donor beads (Packard), 15 μL of 5 mg/ml monoclonal antibodyab179 (this antibody recognizes the portion of the human placentalalkaline phosphatase protein contained in the recombinant EPO-R),protein A-coated acceptor beads (protein A will bind to the ab179antibody; Packard), 112.5 μL of a 1:6.6 dilution of recombinant EPO-R(produced in Chinese Hamster Ovary cells as a fusion protein to aportion of the human placental alkaline phosphatase protein whichcontains the ab179 target epitope) and 607.5 μL of Alphaquest buffer (40mM HEPES, pH 7.4; 1 mM MgCl₂; 0.1% BSA, 0.05% Tween 20). Tap to mix. Add2 μL/well of the biotinylated EPO-R-binding peptide tracer, AF33068 (30nM final concentration). AF33068, an EPO-R binding peptide (see Table 3“Reporter EC50 (pM)”), is made according to the methods described inExample 1.

Centrifuge 1 min to mix. Seal plate with Packard Top Seal and wrap infoil. Incubate overnight at room temperature. After 18 hours read lightemission using an AlphaQuest reader (Packard). Plot light emission vsconcentration of peptide and analyse with Graph Pad or Excel.

The concentration of test peptide that results in a 50% decrease inlight emission, relative to that observed without test peptide, isrecorded as the IC50 [See Table 2: AQ IC50].

4. C/BFU-e Assay

EPO-R signaling stimulates the differentiation of bone marrow stem cellsinto proliferating red blood cell presursors. This assay measures theability of test peptides to stimulate the proliferation anddifferentiation of red blood cell precursors from primary human bonemarrow pluripotent stem cells.

For this assay, serial dilutions of test peptide are made in IMDM medium(Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, orpositive control EPO peptide, are then added to methylcellulose to givea final volume of 1.5 mL. The methylcellulose and peptide mixture isthen vortexed thoroughly. Aliquots (100,000 cells/mL) of human, bonemarrow derived CD34+ cells (Poietics/Cambrex) are thawed. The thawedcells are gently added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50mL tube. Next, 40-50 mL IMDM medium is added gently to cells: the mediumis added drop by drop along the side of the 50 mL tube for the first 10mL, and then the remaining volume of medium is slowly dispensed alongthe side of the tube. The cells are then spun at 900 rpm for 20 min, andthe media removed carefully by gentle aspiration. The cells areresuspended in 1 ml of IMDM medium and the cell density per mL iscounted on hemacytometer slide (10 μL aliquot of cell suspension onslide, and cell density is the average count X 10,000 cells/ml). Thecells are then diluted in IMDM medium to a cell density of 15,000cells/mL. A 100 μL of diluted cells is then added to each 1.5 mL methylcellulose plus peptide sample (final cell concentration in assay mediais 1000 cells/mL), and the mixture is vortexed. Allow the bubbles in themixture to disappear, and then aspirate 1 mL using blunt-end needle. Add0.25 mL aspirated mixture from each sample into each of 4 wells of a24-well plate (Falcon brand). Incubate the plated mixtures at 37° C.under 5% CO₂ in a humid incubator for 14 days. Score for the presence oferythroid colonies using a phase microscope (5×-10× objective, finalmagnification of 100×). The concentration of test peptide at which thenumer of formed colonies is 90% of maximum, relative to that observedwith the EPO positive control, is recorded as the EC90 [See Table 2:C/BFU-e EC90].

5. Radioligand Competitive Binding Assay

An alternative radioligand competition binding assay can also be used tomeasure IC₅₀ values of peptides in this invention. This assay measuresbinding of ¹²⁵-EPO to EPOr. The assay is preferably performed accordingto the following exemplary protocol:

A. Materials Recombinant Human EPO R/Fc Identification: RecombinantHuman EPO R/Fc Chimera Chimera Supplier: R&D Systems (Minneapolis, MN,US) Catalog number: 963-ER Lot number: EOK033071 Storage: 4° C.Iodinated recombinant human Identification:(3[¹²⁵I]iodotyrosyl)Erythropoietin, human Erythropoietin recombinant,high specific activity, 370 kBq, 10 μCi Supplier: Amersham Biosciences(Piscataway, NJ, US) Catalog number: IM219-10 μCi Lot number: Storage:4° C. Protein-G Sepharose Identification: Protein-G Sepharose 4 FastFlow Supplier: Amersham Biosciences (Piscataway, NJ, US) Catalog number17-0618-01 Lot number: Storage: 4° C. Assay Buffer Phosphate BufferedSaline (PBS), pH 7.4, containing 0.1% Bovine Serum Albumin and 0.1%Sodium Azide Storage: 4° C.

B. Determination of Appropriate Receptor Concentration.

One 50 μg vial of lyophilized recombinant EPOr extracellular domainfused to the Fc portion of human IgG1 is reconstituted in 1 mL of assaybuffer. To determine the correct amount of receptor to use in the assay,100 μL serial dilutions of this receptor preparation are combined withapproximately 20,000 cpm in 200 μL of iodinated recombinant humanErythropoietin (¹²⁵I-EPO) in 12×75 mm polypropylene test tubes. Tubesare capped and mixed gently at 4° C. overnight on a LabQuake rotatingshaker. The next day, 50 μL of a 50% slurry of Protein-G Sepharose isadded to each tube. Tubes are then incubated for 2 hours at 4° C.,mixing gently. The tubes are then centrifuged for 15 min at 4000 RPM(3297×G) to pellet the protein-G sepharose. The supernatants arecarefully removed and discarded. After washing 3 times with 1 mL of 4°C. assay buffer, the pellets are counted in a Wallac Wizard gammacounter. Results are then analyzed and the dilution required to reach50% of the maximum binding value is calculated.

C. IC₅₀ Determination for Peptide

To determine the IC₅₀ of AF37702, 100 μL serial dilutions of the peptideare combined with 100 μL of recombinant erythropoietin receptor (100pg/tube) in 12×75 mm polypropylene test tubes. Then 100 μL of iodinatedrecombinant human Erythropoietin (¹²⁵I-EPO) is added to each tube andthe tubes are capped and mixed gently at 4° C. overnight.

The next day, bound ¹²⁵I-EPO is quantitated as described above. Theresults are analyzed and the IC₅₀ value calculated using Graphpad Prismversion 4.0, from GraphPad Software, Inc. (San Diego, Calif.). The assayis repeated two or more times for each peptide tested, for a total of 3or more replicate IC₅₀ determinations. TABLE 2 24/41 In vitro activityassays for peptide dimers Com- Pro- pound Reporter liferation C/BFU-edesig- EC5O EC5O IC50 EC90 nation Peptide dimer (pM) (nM) (pM) (nM)AF35645

. . . . . . 110 2.2 AF35527

150 72 . . . 2.7

Example 4 In Vivo Activity Assays

This example describes various in vivo assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. EPO-R agonist peptide dimers are prepared according to themethods provided in Example 1 or Example 2. The in vivo activity ofthese peptide monomers and dimers is evaluated using a series assays,including a polycythemic exhypoxic mouse bioassay and a reticulocyteassay. These two assays are described in further detail below.

1. Polycythemic Exhypoxic Mouse Bioassay

Test peptides are assayed for in vivo activity in the polycythemicexhypoxic mouse bioassay adapted from the method described by Cotes andBangham (1961), Nature 191: 1065-1067. This assay examines the abilityof a test peptide to function as an EPO mimetic: i.e., to activate EPO-Rand induce new red blood cell synthesis. Red blood cell synthesis isquantitated based upon incorporation of radiolabeled iron intohemoglobin of the synthesized red blood cells.

BDF1 mice are allowed to acclimate to ambient conditions for 7-10 days.Body weights are determined for all animals, and low weight animals (<15grams) are not used. Mice are subjected to successive conditioningcycles in a hypobaric chamber for a total of 14 days. Each 24 hour cycleconsista of 18 hr at 0.40±0.02% atmospheric pressure and 6 hr at ambientpressure. After conditioning the mice are maintained at ambient pressurefor an additional 72 hr prior to dosing.

Test peptides, or recombinant human EPO standards, are diluted inPBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock solutions arefirst solubilized in dimethyl sulfoxide (DMSO). Negative control groupsinclude one group of mice injected with PBS/BSA alone, and one groupinjected with 1% DMSO. Each dose group containa 10 mice. Mice areinjected subcutaneously (scruff of neck) with 0.5 mL of the appropriatesample.

Forty eight hours following sample injection, the mice are administeredan intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), for a doseof approximately 0.75 μCuries/mouse. Mouse body weights are determined24 hr after Fe⁵⁹ administration, and the mice are sacrificed 48 hr afterFe⁵⁹ administration. Blood is collected from each animal by cardiacpuncture and hematocrits are determined (heparin was used as theanticoagulant). Each blood sample (0.2 ml) is analyzed for Fe⁵⁹incorporation using a Packard gamma counter. Non-responder mice (i.e.,those mice with radioactive incorporation less than the negative controlgroup) are eliminated from the appropriate data set. Mice that havehematocrit values less than 53% of the negative control group are alsoeliminated.

Results are derived from sets of 10 animals for each experimental dose.The average amount of radioactivity incorporated [counts per minute(CPM)] into blood samples from each group is calculated.

2. Reticulocyte Assay

Normal BDFI mice are dosed (0.5 mL, injected subcutaneously) on threeconsecutive days with either EPO control or test peptide. At day three,mice are also dosed (0.1 mL, injected intraperitoneally) with irondextran (100 mg/ml). At day five, mice are anesthetized with CO₂ andbled by cardiac puncture. The percent (%) reticulocytes for each bloodsample is determined by thiazole orange staining and flow cytometeranalysis (retic-count program). Hematocrits are manually determined. Thecorrected percent of reticulocytes is determined using the followingformula:% RETIC _(CORRECTED)=% RETIC _(OBSERVED)X(Hematocrit_(INDIVIDUAL)/Hematocrit_(NORMAL))3. Hematological Assay

Normal CD1 mice are dosed with four weekly bolus intravenous injectionsof either EPO positive control, test peptide, or vehicle. A range ofpositive control and test peptide doses, expressed as mg/kg, are testedby varying the active compound concentration in the formulation. Volumesinjected are 5 ml/kg. The vehicle control group is comprised twelveanimals, while 8 animals are in each of the remaining dose groups. Dailyviability and weekly body weights are recorded.

The dosed mice are mice are fasted and then anesthetized with inhaledisoflurane and terminal blood samples are collected via cardiac orabdominal aorta puncture on Day 1 (for vehicle control mice) and on Days15 and 29 (4 mice/group/day). The blood is transferred to Vacutainer®brand tubes. Preferred anticoagulant is ethylenediaminetetraacetic acid(EDTA).

Blood samples are evaluated for endpoints measuring red blood synthesisand physiology such as hematocrit (Hct), hemoglobin (Hgb) and totalerythrocyte count (RBC) using automated clinical analysers well known inthe art (e.g., those made by Coulter, Inc.).

Example 5 Synthesis of EPO-R Agonist Peptide Homodimers of PeptideMonomers Having the Amino Acid Sequence (AcG)GLYACHMGPIT(1-nal)VCQPLRK(SEQ ID NO: 1)

Step 1—Synthesis of peptide monomers: Peptide monomers are synthesizedusing standard Fmoc chemistry on an ABI 431A peptide synthesizer, usingTG-RAM resin (0.18 mmol/g Rapp Polymere, Germany). For the synthesis ofpeptide monomers with an amidated carboxy terminus, the fully assembledpeptide is cleaved from the resin with 82.5% TFA, 5% water, 6.25%anisole, 6.25% ethanedithiol. The deprotected product is filtered fromthe resin and precipitated with diethyl ether. After thorough drying,the product is purified by C18 reverse phase high performance liquidchromatography with a gradient of acetonitrile/water in 0.1%trifluoroacetic acid. The structure of the peptide is confirmed byelectospray mass spectrometry. The peptide is dissolved in a 1:1solution of DMSO:water at a concentration of 1 mg/mL to affect disulfideformation. The product is purified by C18 reverse phase high performanceliquid chromatography with a gradient of acetonitrile/water in 0.1%trifluoroacetic acid. The peptide monomers may be illustrated asfollows:

Step 2—Synthesis of the trifunctional linker: To a solution of diethyliminoacetate (10.0 g, 52.8 mmol) and Boc-beta-alanine (10.0 g, 52.8mmol) in 100 mL of DCM was added diisopropylcarbodiimide (8.0 mL, 51.1mmol) over 10 minutes at room temperture. The reaction mixture warmed to10 degrees during the addition, then cooled back to room temperatureover 20 minutes. The reaction mixture was allowed to stir overnight andthe precepitated diisopropylurea was filtered off. The solvent wasremoved under reduced pressure to afford a gum, and the residuedissolved in ethyl acetate and again filtered to remove the additionalprecipatated urea. The organic phase was placed into a separtory funnel,washed (sat. NaHCO₃, brine, 0.5 N HCl, brine), dried (MgSO₄), filteredand concentrated under reduced pressure to afford the diester product asa colorless oil. The diester was taken up in a 1:1 mixture of MeOH:THF(100 mL) and to this was added water (25 mL), and then NaOH (5 g, 125mmol). The pH was measured to be >10. The reaction mixture was stirredat room temperature for 2 h, and then acidified to pH 1 with 6N HCl. Theaq. Phase was staurated with NaCl and extracted 4 times with ethylacetate. The combined organic phase was washed (brine), dried (MgSO₄),and concentrated under reduced pressure to give a white semi-solid. Thesolid was dissolved in 50 mL of DCM and to this was added 300 mL hexaneto create a white slurry. The solvent was removed under reduced pressureto afford the diacid as a white solid (14.7 g, 91.5% yield for 2 steps).To a solution of the diacid (1 g, 3.29 mmol) in 20 mL of DMF was addedN-hydroxysuccinimide (770 mg, 6.69 mmol) and diisopropylcarbodiimide(1.00 mL, 6.38 mmol) and 4-dimethylaminopyridine (3 mg, 0.02 mmol). Thereaction mixture was stirred overnight and the solvent removed underreduced pressure. The residue was taken up in ethyl acetate and filteredto remove the precpitated urea. The organic phase was placed into asepartory funnel, washed (sat. NaHCO₃, brine, 0.5 N HCl, brine), dried(MgSO₄), filtered and concentrated under reduced pressure to afford thedi-NHS ester product as a white solid (1.12 g, 68% yield).

Step 3—Coupling of the trifunctional linker to the peptide monomers: Forcoupling to the linker, 2 eq peptide is mixed with 1 eq of trifunctionallinker in dry DMF to give clear solution, 5 eq of DIEA is added after 2minutes. The mixture is stirred at ambient temperature for 14 h. Thesolvent is removed under reduced pressure and the crude product isdissolved in 80% TFA in DCM for 30 min to remove the Boc group, followedby purification with C18 reverse phase HPLC. The structure of the dimeris confirmed by clectrospray mass spectrometry. This coupling reactionattaches the linker to the nitrogen atom of the ε-amino group of thelysine residue of each monomer.

Step 4—PEGylation of the Peptide Dimer:

PEGylation via a carbamate bond: The peptide dimer is mixed with anequal amount (mole basis) of activated PEG species (MPEG-NPC from NOFCorp. Japan) in dry DMF to afford a clear solution. After 5 minutes 4 eqof DIEA is added to above solution. The mixture is stirred at ambienttemperature 14 h, followed by purification with C18 reverse phase HPLC.The structure of PEGylated peptide is confirmed by MALDI mass. Thepurified peptide was also subjected to purification via cation ionexchange chromatography as outlined below.

PEGylation via an amide bond: The peptide dimer is mixed with an equalamount (mole basis) of activated PEG species (PEG-SPA-NHS fromShearwater Corp, USA) in dry DMF to afford a clear solution. After 5minutes 10 eq of DIEA is added to above solution. The mixture is stirredat ambient temperature 2 h, followed by purification with C18 reversephase HPLC. The structure of PEGylated peptide was confirmed by MALDImass. The purified peptide was also subjected to purification via cationion exchange chromatography as outlined below.

Step 5: —Ion exchange purification of peptides: Several exchangesupports were surveyed for their ability to separate the abovepeptide-PEG conjugate from unreacted (or hydrolyzed) PEG, in addition totheir ability to retain the starting dimeric peptides. The ion exchangeresin (2-3 g) was loaded into a 1 cm column, followed by conversion tothe sodium form (0.2 N NaOH loaded onto column until elutant was pH 14,ca. 5 column volumes), and than to the hydrogen form (eluted with either0.1 N HCl or 0.1 M HOAc until elutant matched load pH, ca. 5 columnvolumes), followed by washing with 25% ACN/water until pH 6. Either thepeptide prior to conjugation or the peptide-PEG conjugate was dissolvedin 25% ACN/water (10 mg/mL) and the pH adjusted to <3 with TFA, thenloaded on the column. After washing with 2-3 column volumes of 25%ACN/water and collecting 5 mL fractions, the peptide was released fromthe column by elution with 0.1 M NH₄OAc in 25% ACN/water, againcollecting 5 mL fractions. Analysis via HPLC revealed which fractionscontained the desired peptide. Analysis with an EvaporativeLight-Scattering Detector (ELSD) indicated that when the peptide wasretained on the column and was eluted with the NH₄OAc solution(generally between fractions 4 and 10), no non-conjugated PEG wasobserved as a contaminant. When the peptide eluted in the initial washbuffer (generally the first 2 fractions), no separation of desiredPEG-conjugate and excess PEG was observed.

The following columns successfully retained both the peptide and thepeptide-PEG conjugate, and successfully purified the peptide-PEGconjugate from the unconjugates peptide: TABLE 3 Ion Exhange ResinsSupport Source Mono S HR 5/5 strong cation Amersham Biosciences exchangepre-loaded column SE53 Cellulose, microgranular Whatman strong cationexchange support SP Sepharose Fast Flow strong Amersham Biosciencescation exchange support

Example 6 Synthesis of EPO-R Agonist Peptide Homodimers of PeptideMonomers Having the Amino Acid Sequence (AcG)GLYACEMGPIT(1-nal)VCQPLRK(SEQ ID NO: 1)

EPO-R agonist peptide homodimers of peptide monomers having the aminoacid sequence (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) aresynthesized as described in Example 1, except that in Step 1 thesynthesized peptide monomers are: (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K

Where the PEG is attached to the linker via a carbamate linkage, thefinal product of this synthesis may by illustrated structurally asfollows:

Where the PEG is attached to the linker via an amide linkage, the finalproduct of this synthesis may by illustrated structurally as follows:

Example 7 In vitro Activity Assays

This example describes various in vitro assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. The results for these assays demonstrate that the novelpeptides of this invention bind to EPO-R and activate EPO-R signaling.Moreover, the results for these assays show that the novel peptidecompositions exhibit a surprising increase in EPO-R binding affinity andbiological activity compared to EPO mimetic peptides that have beenpreviously described.

EPO-R agonist peptide monomers and dimers are prepared according to themethods provided in Example 1 or Example 2. The potency of these peptidedimers is evaluated using a series of in vitro activity assays,including: a reporter assay, a proliferation assay, a competitivebinding assay, and a C/BFU-e assay. These four assays are described infurther detail below.

The results of these in vitro activity assays are summarized in Table 2.

1. Reporter Assay

This assay is based upon a on a murine pre-B-cell lin derived reportercell, Baf3/EpoR/GCSFR fos/lux. This reporter cell line expresses achimeric receptor comprising the extra-cellular portion of the human EPOreceptor to the intra-cellular portion of the human GCSF receptor. Thiscell line is further transfected with a fos promoter-driven luciferasereporter gene construct. Activation of this chimeric receptor throughaddition of erythropoietic agent results in the expression of theluciferase reporter gene, and therefore the production of light uponaddition of the luciferase substrate luciferin. Thus, the level of EPO-Ractivation in such cells may be quantitated via measurement ofluciferase activity.

The Baf3/EpoRIGCSFR fos/lux cells are cultured in DMEM/F12 medium(Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10%WEHI-3 supernatant (the supernatant from a culture of WEHI-3 cells, ATCC# TIB-68), and penicillin/streptomycin. Approximately 18 h before theassay, cells are starved by transferring them to DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day ofassay, cells are washed once with DMEM/F12 medium supplemented with 10%FBS (no WEHI-3 supernatant), then 1×10⁶ cells/mL are cultured in thepresence of a known concentration of test peptide, or with EPO(R & DSystems Inc., Minneapolis, Minn.) as a positive control, in DMEM/F12medium supplemented with 10% FBS (no WEHI-3 supernatant). Serialdilutions of the test peptide are concurrently tested in this assay.Assay plates are incubated for 4 h at 37° C. in a 5% CO₂ atmosphere,after which luciferin (Steady-Glo; Promega, Madison, Wis.) is added toeach well. Following a 5-minute incubation, light emission is measuredon a Packard Topcount Luminometer (Packard Instrument Co., DownersGrove, Ill.). Light counts are plotted relative to test peptideconcentration and analysed using Graph Pad software. The concentrationof test peptide that results in a half-maximal emission of light isrecorded as the EC50.

2. Proliferation Assay

This assay is based upon a murine pre-B-cell line, BaB, transfected toexpress human EPO-R. Proliferation of the resulting cell line,BaF3/Gal4/Elk/EPOR, is dependent on EPO-R activation. The degree of cellproliferation is quantitated using MTT, where the signal in the MTTassay is proportional to the number of viable cells.

The BaF3/Gal4/Elk/EPOR cells are cultured in spinner flasks in DMEM/F12medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3supernatant (ATCC # TIB-68). Cultured cells are starved overnight, in aspinner flask at a cell density of 1×10⁶ cells/ml, in DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cellsare then washed twice with Dulbecco's PBS (Gibco), and resuspended to adensity of 1×10⁶ cells/ml in DMEM/F12 supplemented with 10% FBS (noWEHI-3 supernatant). 50 μL aliquots (˜50,000 cells) of the cellsuspension are then plated, in triplicate, in 96 well assay plates. 50μL aliquots of dilution series of test EPO mimetic peptides, or 50 μLEPO(R & D Systems Inc., Minneapolis, Minn.) or Aranesp™ (darbepoeitinalpha, an ERO-R agonist commerically available from Amgen) in DMEM/F12media supplemented with 10% FBS (no WEHI-3 supernatant 1) are added tothe 96 well assay plates (final well volume of 100 μL). For example, 12different dilutions may be tested where the final concentration of testpeptide (or control EPO peptide) ranges from 810 pM to 0.0045 pM. Theplated cells are then incubated for 48 h at 37° C. Next, 10 μL of MTT(Roche Diagnostics) is added to each culture dish well, and then allowedto incubate for 4 h. The reaction is then stopped by adding 10%SDS+0.01N HCl. The plates are then incubated overnight at 37° C.Absorbance of each well at a wavelenght of 595 nm is then measured byspectrophotometry. Plots of the absorbance readings versus test peptideconcentration are constructed and the EC50 calculated using Graph Padsoftware. The concentration of test peptide that results in ahalf-maximal absorbance is recorded as the EC50.

3. Competitive Binding Assay

Competitive binding calculations are made using an assay in which alight signal is generated as a function of the proximity of two beads: astreptavidin donor bead bearing a biotinylated EPO-R-binding peptidetracer and an acceptor bead to which is bound EPO-R. Light is generatedby non-radiative energy transfer, during which a singlet oxygen isreleased from a first bead upon illumination, and contact with thereleased singlet oxygen causes the second bead to emit light. These beadsets are commercially available (Packard). Bead proximity is generatedby the binding of the EPO-R-binding peptide tracer to the EPO-R. A testpeptide that competes with the EPO-R-binding peptide tracer for bindingto EPO-R will prevent this binding, causing a decrease in lightemission.

In more detail the method is as follows: Add 4 μL of serial dilutions ofthe test EPO-R agonist peptide, or positive or negative controls, towells of a 384 well plate. Thereafter, add 2 μL/well of receptor/beadcocktail. Receptor bead cocktail consists of: 15 μL of 5 mg/mlstreptavidin donor beads (Packard), 15 μL of 5 mg/ml monoclonal antibodyab179 (this antibody recognizes the portion of the human placentalalkaline phosphatase protein contained in the recombinant EPO-R),protein A-coated acceptor beads (protein A will bind to the ab179antibody; Packard), 112.5 μL of a 1:6.6 dilution of recombinant EPO-R(produced in Chinese Hamster Ovary cells as a fusion protein to aportion of the human placental alkaline phosphatase protein whichcontains the ab179 target epitope) and 607.51 μL of Alphaquest buffer(40 mM HEPES, pH 7.4; 1 mM MgCl₂; 0.1% BSA, 0.05% Tween 20). Tap to mix.Add 2 μL/well of the biotinylated EPO-R-binding peptide tracer, AF33068(30 nM final concentration). AF33068, an EPO-R binding peptide (seeTable 3 “Reporter EC50 (pM)”), is made according to the methodsdescribed in Example 1.

Centrifuge 1 min to mix. Seal plate with Packard Top Seal and wrap infoil. Incubate overnight at room temperature. After 18 hours read lightemission using an AlphaQuest reader (Packard). Plot light emission vsconcentration of peptide and analyse with Graph Pad or Excel.

The concentration of test peptide that results in a 50% decrease inlight emission, relative to that observed without test peptide, isrecorded as the IC50.

4. C/BFU-e Assay

EPO-R signaling stimulates the differentiation of bone marrow stem cellsinto proliferating red blood cell presursors. This assay measures theability of test peptides to stimulate the proliferation anddifferentiation of red blood cell precursors from primary human bonemarrow pluripotent stem cells.

For this assay, serial dilutions of test peptide are made in IMDM medium(Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, orpositive control EPO peptide, are then added to methylcellulose to givea final volume of 1.5 mL. The methylcellulose and peptide mixture isthen vortexed thoroughly. Aliquots (100,000 cells/mL) of human, bonemarrow derived CD34+cells (Poietics/Cambrex) are thawed. The thawedcells are gently added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50mL tube. Next, 40-50 mL IMDM medium is added gently to cells: the mediumis added drop by drop along the side of the 50 mL tube for the first 10mL, and then the remaining volume of medium is slowly dispensed alongthe side of the tube. The cells are then spun at 900 rpm for 20 min, andthe media removed carefully by gentle aspiration. The cells areresuspended in 1 ml of IMDM medium and the cell density per mL iscounted on hemacytometer slide (10 μL aliquot of cell suspension onslide, and cell density is the average count X 10,000 cells/ml). Thecells are then diluted in IMDM medium to a cell density of 15,000cells/mL. A 100 μL of diluted cells is then added to each 1.5 mL methylcellulose plus peptide sample (final cell concentration in assay mediais 1000 cells/mL), and the mixture is vortexed. Allow the bubbles in themixture to disappear, and then aspirate 1 mL using blunt-end needle. Add0.25 mL aspirated mixture from each sample into each of 4 wells of a24-well plate (Falcon brand). Incubate the plated mixtures at 37° C.under 5% CO₂ in a humid incubator for 14 days. Score for the presence oferythroid colonies using a phase microscope (5×-10× objective, finalmagnification of 100×). The concentration of test peptide at which thenumer of formed colonies is 90% of maximum, relative to that observedwith the EPO positive control, is recorded as the EC90 [See Table 2:C/BFU-e EC90]. TABLE 4 In vitro activity assays for peptide dimers Com-Pro- pound Reporter liferation AQ C/BFU-e desig- EC50 EC50 IC50 EC90nation Peptide dimer (pM) (pM) (nM) (nM) AF36205

. . . . . . . . . 6.2

Example 8 In Vivo Activity Assays

This example describes various in, vivo assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. EPO-R agonist peptide monomers and dimers are preparedaccording to the methods provided in Example 1. The in vivo activity ofthese peptide monomers and dimers is evaluated using a series assays,including a polycythemic exhypoxic mouse bioassay and a reticulocyteassay. These two assays are described in further detail below.

1. Polycythemic Exhypoxic Mouse Bioassay

Test peptides are assayed for in vivo activity in the polycythemicexhypoxic mouse bioassay adapted from the method described by Cotes andBangham (1961), Nature 191: 1065-1067. This assay examines the abilityof a test peptide to function as an EPO mimetic: i.e., to activate EPO-Rand induce new red blood cell synthesis. Red blood cell synthesis isquantitated based upon incorporation of radiolabeled iron intohemoglobin of the synthesized red blood cells.

BDF1 mice are allowed to acclimate to ambient conditions for 7-10 days.Body weights are determined for all animals, and low weight animals (<15grams) are not used. Mice are subjected to successive conditioningcycles in a hypobaric chamber for a total of 14 days. Each 24 hour cycleconsista of 18 hr at 0.40±0.02% atmospheric pressure and 6 hr at ambientpressure. After conditioning the mice are maintained at ambient pressurefor an additional 72 hr prior to dosing.

Test peptides, or recombinant human EPO standards, are diluted inPBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock solutions arefirst solubilized in dimethyl sulfoxide (DMSO). Negative control groupsinclude one group of mice injected with PBS/BSA alone, and one groupinjected with 1% DMSO. Each dose group containa 10 mice. Mice areinjected subcutaneously (scruff of neck) with 0.5 mL of the appropriatesample.

Forty eight hours following sample injection, the mice are administeredan intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), for a doseof approximately 0.75 μCuries/mouse. Mouse body weights are determined24 hr after Fe⁵⁹ administration, and the mice are sacrificed 48 hr afterFe⁵⁹ administration. Blood is collected from each animal by cardiacpuncture and hematocrits are determined (heparin was used as theanticoagulant). Each blood sample (0.2 ml) is analyzed for Fe⁵⁹incorporation using a Packard gamma counter. Non-responder mice (i.e.,those mice with radioactive incorporation less than the negative controlgroup) are eliminated from the appropriate data set. Mice that havehematocrit values less than 53% of the negative control group are alsoeliminated.

Results are derived from sets of 10 animals for each experimental dose.The average amount of radioactivity incorporated [counts per minute(CPM)] into blood samples from each group is calculated.

2. Reticulocyte Assay

Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously) on threeconsecutive days with either EPO control or test peptide. At day three,mice are also dosed (0.1 mL, injected intraperitoneally) with irondextran (100 mg/ml). At day five, mice are anesthetized with CO₂ andbled by cardiac puncture. The percent (%) reticulocytes for each bloodsample is determined by thiazole orange staining and flow cytometeranalysis (retic-count program). Hematocrits are manually determined. Thecorrected percent of reticulocytes is determined using the followingformula:% RETIC _(CORRECTED)=% RETIC _(OBSERVED)X(Hematocrit_(INDIVIDUAL)/Hematocrit_(NORMAL))3. Hematological Assay

Normal CD1 mice are dosed with four weekly bolus intravenous injectionsof either EPO positive control, test peptide, or vehicle. A range ofpositive control and test peptide doses, expressed as mg/kg, are testedby varying the active compound concentration in the formulation. Volumesinjected are 5 ml/kg. The vehicle control group is comprised twelveanimals, while 8 animals are in each of the remaining dose groups. Dailyviability and weekly body weights are recorded.

The dosed mice are mice are fasted and then anesthetized with inhaledisoflurane and terminal blood samples are collected via cardiac orabdominal aorta puncture on Day 1 (for vehicle control mice) and on Days15 and 29 (4 mice/group/day). The blood is transferred to Vacutainer™brand tubes. Preferred anticoagulant is ethylenediaminetetraacetic acid(EDTA).

Blood samples are evaluated for endpoints measuring red blood synthesisand physiology such as hematocrit (Hct), hemoglobin (Hgb) and totalerythrocyte count (RBC) using automated clinical analysers well known inthe art (e.g., those made by Coulter, Inc.).

Example 9 Synthesis of EPO-R Agonist Peptide Homodimers of PeptideMonomers Having the Amino Acid Sequence (AcG)GLYACEMGPIT(1-nal)VCQPLRK(SEQ ID NO: 1)

Step 1—Synthesis of peptide monomers: Peptide monomers are synthesizedusing standard Fmoc chemistry on an ABI 431A peptide synthesizer, usingTG-RAM resin (0.18 mmol/g Rapp Polymere, Germany). For the synthesis ofpeptide monomers with an amidated carboxy terminus, the fully assembledpeptide is cleaved from the resin with 82.5% TFA, 5% water, 6.25%anisole, 6.25% ethanedithiol. The deprotected product is filtered fromthe resin and precipitated with diethyl ether. After thorough drying theproduct is purified by C18 reverse phase high performance liquidchromatography with a gradient of acetonitrile/water in 0.1%trifluoroacetic acid. The structure of the peptide is confirmed byelectospray mass spectrometry. The peptide monomers may be illustratedas follows: (AcG)GLYACHMGPIT(1-nal)VCQPLRK-NH₂

Step 2—Synthesis of the Trifunctional Linker:

To a solution of diethyl iminoacetate (10.0 g, 52.8 mmol) andBoc-beta-alanine (10.0 g, 52.8 mmol) in 100 mL of DCM was addeddiisopropylcarbodiimide (8.0 mL, 51.1 mmol) over 10 minutes at roomtemperture. The reaction mixture warmed to 10 degrees during theaddition, then cooled back to room temperature over 20 minutes. Thereaction mixture was allowed to stir overnight and the precepitateddiisopropylurea was filtered off. The solvent was removed under reducedpressure to afford a gum, and the residue dissolved in ethyl acetate andagain filtered to remove the additional precipatated urea. The organicphase was placed into a separtory funnel, washed (sat. NaHCO₃, brine,0.5 N HCl, brine), dried (MgSO₄), filtered and concentrated underreduced pressure to afford the diester product as a colorless oil. Thediester was taken up in a 1:1 mixture of MeOH:THF (100 mL) and to thiswas added water (25 mL), and then NaOH (5 g, 125 mmol). The pH wasmeasured to be >10. The reaction mixture was stirred at room temperaturefor 2 h, and then acidified to pH 1 with 6N HCl. The aq. Phase wassaturated with NaCl and extracted 4 times with ethyl acetate. Thecombined organic phase was washed (brine), dried (MgSO₄), andconcentrated under reduced pressure to give a white semi-solid. Thesolid was dissolved in 50 mL of DCM and to this was added 300 mL hexaneto create a white slurry. The solvent was removed under reduced pressureto afford the diacid as a white solid (14.7 g, 91.5% yield for 2 steps).To a solution of the diacid (1 g, 3.29 mmol) in 20 mL of DMF was addedN-hydroxysuccinimide (770 mg, 6.69 mmol) and diisopropylcarbodiimide(1.00 mL, 6.38 mmol) and 4-dimethylaminopyridine (3 mg, 0.02 mmol). Thereaction mixture was stirred overnight and the solvent removed underreduced pressure. The residue was taken up in ethyl acetate and filteredto remove the precipitated urea. The organic phase was placed into asepartory funnel, washed (sat. NaHCO₃, brine, 0.5 N HCl, brine), dried(MgSO₄), filtered and concentrated under reduced pressure to afford thedi-NHS ester product as a white solid (1.12 g, 68% yield).

Step 3—Coupling of the Trifunctional Linker to the Peptide Monomers:

For coupling to the linker, 2 eq peptide is mixed with 1 eq oftrifunctional linker in dry DMF to give a clear solution, and 5 eq ofDIEA is added after 2 minutes. The mixture is stirred at ambienttemperature for 14 h. The solvent is removed under reduced pressure andthe crude product is dissolved in 80% TFA in DCM for 30 min to removethe Boc group, followed by purification with C18 reverse phase HPLC. Thestructure of the dimer is confirmed by electrospray mass spectrometry.This coupling reaction attaches the linker to the nitrogen atom of theε-amino group of the lysine residue of each monomer.

Step 4—Synthesis of PEG moiety comprising two linear PEGS chains linkedby Lysine mPEG2-Lysinol-NPC

Lysinol, which may be obtained commercially, is treated with an excessof mPEG2-NPC to obtain MPEG2-lysinol, which is then reacted with NPC toform mPEG2-lysinol-NPC.

mPEG2-Lys-NAS

This product may be obtained commercially, for example, from theMolecular Engineering catalog (2003) of Nektar Therapeutics (490Discovery Drive, Huntsville, Ala. 35806), item no. 2Z3X0T01.

Step 5—PEGylation of the Peptide Dimer:

PEGylation via a carbamate bond:

The peptide dimer and the PEG species (mPEG₂-Lysinol-NPC) are mixed in a1:2 molar ratio in dry DMF to afford a clear solution. After 5 minutes 4eq of DIEA is added to above solution. The mixture is stirred at ambienttemperature 14 h, followed by purification with C18 reverse phase HPLC.The structure of PEGylated peptide is confirmed by MALDI mass. Thepurified peptide was also subjected to purification via cation ionexchange chromatography as outlined below.

PEGylation via an Amide Bond:

The peptide dimer and PEG species (mPEG₂-Lys-NHS from Shearwater Corp,USA) are mixed in a 1:2 molar ratio in dry DMF to afford a clearsolution. After 5 minutes 10 eq of DIEA is added to above solution. Themixture is stirred at ambient temperature 2 h, followed by purificationwith C18 reverse phase HPLC. The structure of PEGylated peptide wasconfirmed by MALDI mass. The purified peptide was also subjected topurification via cation ion exchange chromatography as outlined below.

Step 6: —Ion exchange purification of peptides: Several exchangesupports were surveyed for their ability to separate the abovepeptide-PEG conjugate from unreacted (or hydrolyzed) PEG, in addition totheir ability to retain the starting dimeric peptides. The ion exchangeresin (2-3 g) was loaded into a 1 cm column, followed by conversion tothe sodium form (0.2 N NaOH loaded onto column until elutant was pH 14,ca. 5 column volumes), and than to the hydrogen form (eluted with either0.1 N HCl or 0.1 M HOAc until elutant matched load pH, ca. 5 columnvolumes), followed by washing with 25% ACN/water until pH 6. Either thepeptide prior to conjugation or the peptide-PEG conjugate was dissolvedin 25% ACN/water (10 mg/mL) and the pH adjusted to <3 with TFA, thenloaded on the column. After washing with 2-3 column volumes of 25%ACN/water and collecting 5 mL fractions, the peptide was released fromthe column by elution with 0.1 M NH₄OAc in 25% ACN/water, againcollecting 5 mL fractions. Analysis via HPLC revealed which fractionscontained the desired peptide. Analysis with an EvaporativeLight-Scattering Detector (ELSD) indicated that when the peptide wasretained on the column and was eluted with the NH₄OAc solution(generally between fractions 4 and 10), no non-conjugated PEG wasobserved as a contaminant. When the peptide eluted in the initial washbuffer (generally the first 2 fractions), no separation of desiredPEG-conjugate and excess PEG was observed.

The following columns successfully retained both the peptide and thepeptide-PEG conjugate, and successfully purified the peptide-PEGconjugate from the unconjugates peptide: TABLE 5 Ion Exhange ResinsSupport Source Mono S HR 5/5 strong cation Amersham Biosciences exchangepre-loaded column SE53 Cellulose, microgranular Whatman strong cationexchange support SP Sepharose Fast Flow strong Amersham Biosciencescation exchange support

Example 10 Synthesis of EPO-R Agonist Peptide Homodimers of PeptideMonomers Having the Amino Acid Sequence(AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 1)

EPO-R agonist peptide homodimers of peptide monomers having the aminoacid sequence (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K (SEQ ID NO: 2) aresynthesized as described in Example 1, except that in Step 1 thesynthesized peptide monomers are: (AcG)GLYACHMGPIT(1-nal)VCQPLR(MeG)K

Where the PEG is attached to the spacer via carbamate linkages, thefinal product of this synthesis may by illustrated structurally asfollows:

Where the PEG is attached to the spacer via amide linkages, the finalproduct of this synthesis may by illustrated structurally as follows:

Example 11 In vitro Activity Assays

This example describes various in vitro assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. The results for these assays demonstrate that the novelpeptides of this invention bind to EPO-R and activate EPO-R signaling.Moreover, the results for these assays show that the novel peptidecompositions exhibit a surprising increase in EPO-R binding affinity andbiological activity compared to EPO mimetic peptides that have beenpreviously described.

EPO-R agonist peptide monomers and dimers are prepared according to themethods provided in Example 1 or Example 2. The potency of these peptidedimers is evaluated using a series of in vitro activity assays,including: a reporter assay, a proliferation assay, a competitivebinding assay, and a C/BFU-e assay. These four assays are described infurther detail below.

The results of these in vitro activity assays are summarized in Table 2.

1. Reporter Assay

This assay is based upon a on a murine pre-B-cell lin derived reportercell, Baf3/EpoR/GCSFR fos/lux. This reporter cell line expresses achimeric receptor comprising the extra-cellular portion of the human EPOreceptor to the intra-cellular portion of the human GCSF receptor. Thiscell line is further transfected with a fos promoter-driven luciferasereporter gene construct. Activation of this chimeric receptor throughaddition of erythropoietic agent results in the expression of theluciferase reporter gene, and therefore the production of light uponaddition of the luciferase substrate luciferin. Thus, the level of EPO-Ractivation in such cells may be quantitated via measurement ofluciferase activity.

The Baf3/EpoR/GCSFR fos/lux cells are cultured in DMEM/F12 medium(Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone), 10%WEHI-3 supernatant (the supernatant from a culture of WEHI-3 cells, ATCC# TIB-68), and penicillin/streptomycin. Approximately 18 h before theassay, cells are starved by transferring them to DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. On the day ofassay, cells are washed once with DMEM/F12 medium supplemented with 10%FBS (no WEHI-3 supernatant), then 1×10⁶ cells/mL are cultured in thepresence of a known concentration of test peptide, or with EPO(R & DSystems Inc., Minneapolis, Minn.) as a positive control, in DMEM/F12medium supplemented with 10% FBS (no WEHI-3 supernatant). Serialdilutions of the test peptide are concurrently tested in this assay.Assay plates are incubated for 4 h at 37° C. in a 5% CO₂ atmosphere,after which luciferin (Steady-Glo; Promega, Madison, Wis.) is added toeach well. Following a 5-minute incubation, light emission is measuredon a Packard Topcount Luminometer (Packard Instrument Co., DownersGrove, Ill.). Light counts are plotted relative to test peptideconcentration and analysed using Graph Pad software. The concentrationof test peptide that results in a half-maximal emission of light isrecorded as the EC50.

2. Proliferation Assay

This assay is based upon a murine pre-B-cell line, Baf3, transfected toexpress human EPO-R. Proliferation of the resulting cell line,BaF3/Gal4/Elk/EPOR, is dependent on EPO-R activation. The degree of cellproliferation is quantitated using MTT, where the signal in the MTTassay is proportional to the number of viable cells.

The BaF3/Gal4/Elk/EPOR cells are cultured in spinner flasks in DMEM/F12medium (Gibco) supplemented with 10% FBS (Hyclone) and 2% WEHI-3supernatant (ATCC # TIB-68). Cultured cells are starved overnight, in aspinner flask at a cell density of 1×10⁶ cells/ml, in DMEM/F12 mediumsupplemented with 10% FBS and 0.1% WEHI-3 supernatant. The starved cellsare then washed twice with Dulbecco's PBS (Gibco), and resuspended to adensity of 1×10⁶ cells/ml in DMEM/F12 supplemented with 10% FBS (noWEHI-3 supernatant). 50 μL aliquots (50,000 cells) of the cellsuspension are then plated, in triplicate, in 96 well assay plates. 50μL aliquots of dilution series of test EPO mimetic peptides, or 50 μLEPO(R & D Systems Inc., Minneapolis, Minn.) or Aranesp™ (darbepoeitinalpha, an ERO-R agonist commercially available from Amgen) in DMEM/F12media supplemented with 10% FBS (no WEHI-3 supernatant 1) are added tothe 96 well assay plates (final well volume of 100 μL). For example, 12different dilutions may be tested where the final concentration of testpeptide (or control EPO peptide) ranges from 810 pM to 0.0045 pM. Theplated cells are then incubated for 48 h at 37° C. Next, 10 μL of MTT(Roche Diagnostics) is added to each culture dish well, and then allowedto incubate for 4 h. The reaction is then stopped by adding 10%SDS+0.01N HCl. The plates are then incubated overnight at 37° C.Absorbance of each well at a wavelength of 595 nm is then measured byspectrophotometry. Plots of the absorbance readings versus test peptideconcentration are constructed and the EC50 calculated using Graph Padsoftware. The concentration of test peptide that results in ahalf-maximal absorbance is recorded as the EC50.

3. Competitive Binding Assay

Competitive binding calculations are made using an assay in which alight signal is generated as a function of the proximity of two beads: astreptavidin donor bead bearing a biotinylated EPO-R-binding peptidetracer and an acceptor bead to which is bound EPO-R. Light is generatedby non-radiative energy transfer, during which a singlet oxygen isreleased from a first bead upon illumination, and contact with thereleased singlet oxygen causes the second bead to emit light. These beadsets are commercially available (Packard). Bead proximity is generatedby the binding of the EPO-R-binding peptide tracer to the EPO-R. A testpeptide that competes with the EPO-R-binding peptide tracer for bindingto EPO-R will prevent this binding, causing a decrease in lightemission.

In more detail the method is as follows: Add 4 μL of serial dilutions ofthe test EPO-R agonist peptide, or positive or negative controls, towells of a 384 well plate. Thereafter, add 2 μL/well of receptor/beadcocktail. Receptor bead cocktail consists of: 15 μL of 5 mg/mlstreptavidin donor beads (Packard), 15 μL of 5 mg/ml monoclonal antibodyab179 (this antibody recognizes the portion of the human placentalalkaline phosphatase protein contained in the recombinant EPO-R),protein A-coated acceptor beads (protein A will bind to the ab179antibody; Packard), 112.5 μL of a 1:6.6 dilution of recombinant EPO-R(produced in Chinese Hamster Ovary cells as a fusion protein to aportion of the human placental alkaline phosphatase protein whichcontains the ab179 target epitope) and 607.5 μL of Alphaquest buffer (40mM HEPES, pH 7.4; 1 mM MgCl₂; 0.1% BSA, 0.05% Tween 20). Tap to mix. Add2 μL/well of the biotinylated EPO-R-binding peptide tracer, AF33068 (30nM final concentration). AF33068, an EPO-R binding peptide (see Table 3“Reporter EC50 (pM)”), is made according to the methods described inExample 1.

Centrifuge 1 min to mix. Seal plate with Packard Top Seal and wrap infoil. Incubate overnight at room temperature. After 18 hours read lightemission using an AlphaQuest reader (Packard). Plot light emission vsconcentration of peptide and analyse with Graph Pad or Excel.

The concentration of test peptide that results in a 50% decrease inlight emission, relative to that observed without test peptide, isrecorded as the IC50.

4. C/BFU-e Assay

EPO-R signaling stimulates the differentiation of bone marrow stem cellsinto proliferating red blood cell precursors. This assay measures theability of test peptides to stimulate the proliferation anddifferentiation of red blood cell precursors from primary human bonemarrow pluripotent stem cells.

For this assay, serial dilutions of test peptide are made in IMDM medium(Gibco) supplemented with 10% FBS (Hyclone). These serial dilutions, orpositive control EPO peptide, are then added to methylcellulose to givea final volume of 1.5 mL. The methylcellulose and peptide mixture isthen vortexed thoroughly. Aliquots (100,000 cells/mL) of human, bonemarrow derived CD34+cells (Poietics/Cambrex) are thawed. The thawedcells are gently added to 0.1 mL of 1 mg/ml DNAse (Stem Cells) in a 50mL tube. Next, 40-50 mL IMDM medium is added gently to cells: the mediumis added drop by drop along the side of the 50 mL tube for the first 10mL, and then the remaining volume of medium is slowly dispensed alongthe side of the tube. The cells are then spun at 900 rpm for 20 min, andthe media removed carefully by gentle aspiration. The cells areresuspended in 1 ml of IMDM medium and the cell density per mL iscounted on hemacytometer slide (10 μL aliquot of cell suspension onslide, and cell density is the average count X 10,000 cells/ml). Thecells are then diluted in IMDM medium to a cell density of 15,000cells/mL. A 100 μL of diluted cells is then added to each 1.5 mL methylcellulose plus peptide sample (final cell concentration in assay mediais 1000 cells/mL), and the mixture is vortexed. Allow the bubbles in themixture to disappear, and then aspirate 1 mL using blunt-end needle. Add0.25 mL aspirated mixture from each sample into each of 4 wells of a24-well plate (Falcon brand). Incubate the plated mixtures at 37° C.under 5% CO₂ in a humid incubator for 14 days. Score for the presence oferythroid colonies using a phase microscope (5×-10× objective, finalmagnification of 100×). The concentration of test peptide at which thenumber of formed colonies is 90% of maximum, relative to that observedwith the EPO positive control, is recorded as the EC90 [See Table 2:C/BFU-e EC90]. TABLE 6 In vitro activity assays for peptide dimers Com-Re- pound porter desig- EC5O nation Peptide dimer (pM) AF37702

195 Pro- Radio Com- lifer- li- C/ pound ation gand BFU-e desig- EC5OIC50 EC9O nation (pM) (pM) (nm) AF37702 165 111 3

Example 12 In Vivo Activity Assays

This example describes various in vivo assays that are useful inevaluating the activity and potency of EPO-R agonist peptides of theinvention. EPO-R agonist peptide monomers and dimers are preparedaccording to the methods provided in Example 1. The in vivo activity ofthese peptide monomers and dimers is evaluated using a series assays,including a polycythemic exhypoxic mouse bioassay and a reticulocyteassay. These two assays are described in further detail below.

1. Polycythemic Exhypoxic Mouse Bioassay

Test peptides are assayed for in vivo activity in the polycythemicexhypoxic mouse bioassay adapted from the method described by Cotes andBangham (1961), Nature 191: 1065-1067. This assay examines the abilityof a test peptide to function as an EPO mimetic: i.e., to activate EPO-Rand induce new red blood cell synthesis. Red blood cell synthesis isquantitated based upon incorporation of radiolabeled iron intohemoglobin of the synthesized red blood cells.

BDF1 mice are allowed to acclimate to ambient conditions for 7-10 days.Body weights are determined for all animals, and low weight animals (<15grams) are not used. Mice are subjected to successive conditioningcycles in a hypobaric chamber for a total of 14 days. Each 24 hour cycleconsists of 18 hr at 0.40±0.02% atmospheric pressure and 6 hr at ambientpressure. After conditioning the mice are maintained at ambient pressurefor an additional 72 hr prior to dosing.

Test peptides, or recombinant human EPO standards, are diluted inPBS+0.1% BSA vehicle (PBS/BSA). Peptide monomer stock solutions arefirst solubilized in dimethyl sulfoxide (DMSO). Negative control groupsinclude one group of mice injected with PBS/BSA alone, and one groupinjected with 1% DMSO. Each dose group contains 10 mice. Mice areinjected subcutaneously (scruff of neck) with 0.5 mL of the appropriatesample.

Forty eight hours following sample injection, the mice are administeredan intraperitoneal injection of 0.2 ml of Fe⁵⁹ (Dupont, NEN), for a doseof approximately 0.75 μCuries/mouse. Mouse body weights are determined24 hr after Fe⁵⁹ administration, and the mice are sacrificed 48 hr afterFe⁵⁹ administration. Blood is collected from each animal by cardiacpuncture and hematocrits are determined (heparin was used as theanticoagulant). Each blood sample (0.2 ml) is analyzed for Fe⁵⁹incorporation using a Packard gamma counter. Non-responder mice (i.e.,those mice with radioactive incorporation less than the negative controlgroup) are eliminated from the appropriate data set. Mice that havehematocrit values less than 53% of the negative control group are alsoeliminated.

Results are derived from sets of 10 animals for each experimental dose.The average amount of radioactivity incorporated [counts per minute(CPM)] into blood samples from each group is calculated.

2. Reticulocyte Assay

Normal BDF1 mice are dosed (0.5 mL, injected subcutaneously) on threeconsecutive days with either EPO control or test peptide. At day three,mice are also dosed (0.1 mL, injected intraperitoneally) with irondextran (100 mg/ml). At day five, mice are anesthetized with CO₂ andbled by cardiac puncture. The percent (%) reticulocytes for each bloodsample is determined by thiazole orange staining and flow cytometeranalysis (retic-count program). Hematocrits are manually determined. Thecorrected percent of reticulocytes is determined using the followingformula:% RETIC _(CORRECTED)=% RETIC _(OBSERVED)X(Hematocrit_(INDIVIDUAL)/Hematocrit_(NORMAL))3. Hematological Assay

Normal CD1 mice are dosed with four weekly bolus intravenous injectionsof either EPO positive control, test peptide, or vehicle. A range ofpositive control and test peptide doses, expressed as mg/kg, are testedby varying the active compound concentration in the formulation. Volumesinjected are 5 ml/kg. The vehicle control group is comprised twelveanimals, while 8 animals are in each of the remaining dose groups. Dailyviability and weekly body weights are recorded.

The dosed mice are mice are fasted and then anesthetized with inhaledisoflurane and terminal blood samples are collected via cardiac orabdominal aorta puncture on Day 1 (for vehicle control mice) and on Days15 and 29 (4 mice/group/day). The blood is transferred to Vacutainer®brand tubes. Preferred anticoagulant is ethylenediaminetetraacetic acid(EDTA).

Blood samples are evaluated for endpoints measuring red blood synthesisand physiology such as hematocrit (Hct), hemoglobin (Hgb) and totalerythrocyte count (RBC) using automated clinical analyzers well known inthe art (e.g., those made by Coulter, Inc.).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Numerous references, including patents, patent applications, and variouspublications are cited and discussed in the description of thisinvention. The citation and/or discussion of such references is providedmerely to clarify the description of the present invention and is not anadmission that any such reference is “prior art” to the presentinvention. All references cited and discussed in this specification areincorporated herein by reference in their entirety and to the sameextent as if each reference was individually incorporated by reference.

1. A compound that binds to and activates the erythropoietin receptor(EPO-R), which compound comprises a peptide dimer having the formula:

wherein (i) in each peptide monomer of the peptide dimer, each aminoacid is indicated by standard one letter abbreviation, AcG isN-acetylglycine, and 1-nal is 1-naphthylalanine; (ii) each peptidemonomer of the peptide dimer contains an intramolecular disulfide bondbetween the two cysteine (C) residues of each monomer (iii) [“PEG”]comprises at least one linear polyethylene glycol (PEG) moiety, each PEGmoiety having a molecular weight of about 20,000 to about 40,000Daltons.
 2. A compound according to claim 1, wherein each PEG has amolecular weight of about 30,000 Daltons.
 3. A method for treating apatient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 1. 4. A method according toclaim 3 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 5. A methodaccording to claim 3, wherein each PEG has a molecular weight of about30,000 Daltons.
 6. A pharmaceutical composition comprising the compoundof claim 1 and a pharmaceutically acceptable carrier.
 7. Apharmaceutical composition according to claim 6, wherein each PEG has amolecular weight of about 30,000 Daltons.
 8. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

where (i) in each peptide monomer of the peptide dimer, each amino acidis indicated by standard one letter abbreviation, AcG isN-acetylglycine, 1-nal is 1-naphthylalanine, and MeG is N-methylglycine;(ii) each peptide monomer of the peptide dimer contains anintramolecular disulfide bond between the two cysteine (C) residues ofeach monomer (iii) PEG comprises a linear unbranched polyethylene glycolmolecule having a molecular weight of about 20,000 to about 40,000Daltons.
 9. A compound according to claim 8, wherein the PEG has amolecular weight of about 30,000 Daltons.
 10. A method for treating apatient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 8. 11. A method according toclaim 10 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 12. A methodaccording to claim 10, wherein the PEG has a molecular weight of about30,000 Daltons.
 13. A pharmaceutical composition comprising the compoundof claim 8 and a pharmaceutically acceptable carrier.
 14. Apharmaceutical composition according to claim 13, wherein the PEG has amolecular weight of about 30,000 Daltons.
 15. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

where (i) in each peptide monomer of the peptide dimer, each amino acidis indicated by standard one letter abbreviation, AcG isN-acetylglycine, and 1-nal is 1-naphthylalanine; (ii) each peptidemonomer of the peptide dimer contains an intramolecular disulfide bondbetween the two cysteine (C) residues of each monomer (iii) PEGcomprises a linear unbranched polyethylene glycol molecule having amolecular weight of about 20,000 to about 40,000 Daltons.
 16. A compoundaccording to claim 15, wherein the PEG has a molecular weight of about30,000 Daltons.
 17. A method for treating a patient having a disordercharacterized by a deficiency of erythropoietin or a low or defectivered blood cell population, which method comprises administering to thepatient a therapeutically effective amount of the compound of claim 15.18. A method according to claim 17 wherein the disorder is selected fromthe group consisting of end stage renal failure or dialysis; anemiaassociated with AIDS, auto immune disease or a malignancy;beta-thalassemia; cystic fibrosis; early anemia of prematurity; anemiaassociated with chronic inflammatory disease; spinal cord injury; acuteblood loss; aging; and neoplastic disease states accompanied by abnormalerythropoiesis.
 19. A method according to claim 17, wherein the PEG hasa molecular weight of about 30,000 Daltons.
 20. A pharmaceuticalcomposition comprising the compound of claim 15 and a pharmaceuticallyacceptable carrier.
 21. A pharmaceutical composition according to claim20, wherein the PEG has a molecular weight of about 30,000 Daltons. 22.A compound that binds to and activates the erythropoietin receptor(EPO-R), which compound comprises a peptide dimer having the formula:

where (i) in each peptide monomer of the peptide dimer, each amino acidis indicated by standard one letter abbreviation, AcG isN-acetylglycine, 1-nal is 1-naphthylalanine, and MeG is N-methylglycine;(ii) each peptide monomer of the peptide dimer contains anintramolecular disulfide bond between the two cysteine (C) residues ofeach monomer (iii) PEG comprises a linear unbranched polyethylene glycolmolecule having a molecular weight of about 20,000 to about 40,000Daltons.
 23. A compound according to claim 22, wherein the PEG has amolecular weight of about 30,000 Daltons.
 24. A method for treating apatient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 22. 25. A method according toclaim 24 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 26. A methodaccording to claim 24, wherein the PEG has a molecular weight of about30,000 Daltons.
 27. A pharmaceutical composition comprising the compoundof claim 22 and a pharmaceutically acceptable carrier.
 28. Apharmaceutical composition according to claim 27, wherein the PEG has amolecular weight of about 30,000 Daltons.
 29. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

where (i) in each peptide monomer of the peptide dimer, each amino acidis indicated by standard one letter abbreviation, AcG isN-acetylglycine, 1-nal is 1-naphthylalanine, and MeG is N-methylglycine;(ii) each peptide monomer of the peptide dimer contains anintramolecular disulfide bond between the two cysteine (C) residues ofeach monomer (iii) PEG comprises a linear unbranched polyethylene glycolmolecule having a molecular weight of about 20,000 to about 40,000Daltons.
 30. A compound according to claim 29, wherein the PEG has amolecular weight of about 30,000 Daltons.
 31. A method for treating apatient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 29. 32. A method according toclaim 31 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 33. A methodaccording to claim 31, wherein the PEG has a molecular weight of about30,000 Daltons.
 34. A pharmaceutical composition comprising the compoundof claim 29 and a pharmaceutically acceptable carrier.
 35. Apharmaceutical composition according to claim 34, wherein the PEG has amolecular weight of about 30,000 Daltons.
 36. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

where (i) in each peptide monomer of the peptide dimer, each amino acidis indicated by standard one letter abbreviation, AcG isN-acetylglycine, 1-nal is 1-naphthylalanine, and MeG is N-methylglycine;(ii) each peptide monomer of the peptide dimer contains anintramolecular disulfide bond between the two cysteine (C) residues ofeach monomer (iii) PEG comprises a linear unbranched polyethylene glycolmolecule having a molecular weight of about 20,000 to about 40,000Daltons.
 37. A compound according to claim 36, wherein the PEG has amolecular weight of about 20,000 Daltons.
 38. A method for treating apatient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 36. 39. A method according toclaim 38 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 40. A methodaccording to claim 38, wherein the PEG has a molecular weight of about20,000 Daltons.
 41. A pharmaceutical composition comprising the compoundof claim 36 and a pharmaceutically acceptable carrier.
 42. Apharmaceutical composition according to claim 41, wherein the PEG has amolecular weight of about 20,000 Daltons.
 43. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

wherein (i) in each peptide monomer of the peptide dimer, each aminoacid is indicated by standard one letter abbreviation, AcG isN-acetylglycine, and 1-nal is 1-naphthylalanine; (ii) each peptidemonomer of the peptide dimer contains an intramolecular disulfide bondbetween the two cysteine (C) residues of each monomer (iii) [“PEG”]comprises at least two linear polyethylene glycol (PEG) moieties linkedat a single point of attachment and having a combined molecular weightof about 10,000 to about 60,000 Daltons.
 44. A compound according toclaim 43, wherein each PEG has a molecular weight of about 20,000Daltons.
 45. A method for treating a patient having a disordercharacterized by a deficiency of erythropoietin or a low or defectivered blood cell population, which method comprises administering to thepatient a therapeutically effective amount of the compound of claim 43.46. A method according to claim 45 wherein the disorder is selected fromthe group consisting of end stage renal failure or dialysis; anemiaassociated with AIDS, auto immune disease or a malignancy;beta-thalassemia; cystic fibrosis; early anemia of prematurity; anemiaassociated with chronic inflammatory disease; spinal cord injury; acuteblood loss; aging; and neoplastic disease states accompanied by abnormalerythropoiesis.
 47. A method according to claim 45, wherein each PEG hasa molecular weight of about 20,000 Daltons.
 48. A pharmaceuticalcomposition comprising the compound of claim 43 and a pharmaceuticallyacceptable carrier.
 49. A pharmaceutical composition according to claim48, wherein each PEG has a molecular weight of about 20,000 Daltons. 50.A compound that binds to and activates the erythropoietin receptor(EPO-R), which compound comprises a peptide dimer having the formula:

where (i) in each peptide monomer of the peptide dimer, each amino acidis indicated by standard one letter abbreviation, AcG isN-acetylglycine, 1-nal is 1-naphthylalanine, and MeG is N-methylglycine;(ii) each peptide monomer of the peptide dimer contains anintramolecular disulfide bond between the two cysteine (C) residues ofeach monomer (iii) PEG comprises two linear polyethylene glycol (PEG)moieties having a combined molecular weight of about 10,000 to about30,000 Daltons.
 51. A compound according to claim 50, wherein each PEGhas a molecular weight of about 20,000 Daltons.
 52. A method fortreating a patient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 50. 53. A method according toclaim 52 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 54. A methodaccording to claim 52, wherein each PEG has a molecular weight of about20,000 Daltons.
 55. A pharmaceutical composition comprising the compoundof claim 50 and a pharmaceutically acceptable carrier.
 56. Apharmaceutical composition according to claim 55, wherein each PEG has amolecular weight of about 20,000 Daltons.
 57. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

wherein (i) in each peptide monomer of the peptide dimer, each aminoacid is indicated by standard one letter abbreviation, AcG isN-acetylglycine, and 1-nal is 1-naphthylalanine; (ii) each peptidemonomer of the peptide dimer contains an intramolecular disulfide bondbetween the two cysteine (C) residues of each monomer (iii) PEGcomprises two linear polyethylene glycol (PEG) moieties having acombined molecular weight of about 10,000 to about 30,000 Daltons.
 58. Acompound according to claim 57, wherein the PEG has a molecular weightof about 20,000 Daltons.
 59. A method for treating a patient having adisorder characterized by a deficiency of erythropoietin or a low ordefective red blood cell population, which method comprisesadministering to the patient a therapeutically effective amount of thecompound of claim
 57. 60. A method according to claim 59 wherein thedisorder is selected from the group consisting of end stage renalfailure or dialysis; anemia associated with AIDS, auto immune disease ora malignancy; beta-thalassemia; cystic fibrosis; early anemia ofprematurity; anemia associated with chronic inflammatory disease; spinalcord injury; acute blood loss; aging; and neoplastic disease statesaccompanied by abnormal erythropoiesis.
 61. A method according to claim59, wherein each PEG has a molecular weight of about 20,000 Daltons. 62.A pharmaceutical composition comprising the compound of claim 57 and apharmaceutically acceptable carrier.
 63. A pharmaceutical compositionaccording to claim 62, wherein each PEG has a molecular weight of about20,000 Daltons.
 64. A compound that binds to and activates theerythropoietin receptor (EPO-R), which compound comprises a peptidedimer having the formula:

wherein (i) in each peptide monomer of the peptide dimer, each aminoacid is indicated by standard one letter abbreviation, AcG isN-acetylglycine, 1-nal is 1-naphthylalanine, and MeG is N-methylglycine;(ii) each peptide monomer of the peptide dimer contains anintramolecular disulfide bond between the two cysteine (C) residues ofeach monomer (iii) PEG comprises two linear polyethylene glycol (PEG)moieties having a combined molecular weight of about 10,000 to about30,000 Daltons.
 65. A compound according to claim 64, wherein each PEGhas a molecular weight of about 20,000 Daltons.
 66. A method fortreating a patient having a disorder characterized by a deficiency oferythropoietin or a low or defective red blood cell population, whichmethod comprises administering to the patient a therapeuticallyeffective amount of the compound of claim
 64. 67. A method according toclaim 66 wherein the disorder is selected from the group consisting ofend stage renal failure or dialysis; anemia associated with AIDS, autoimmune disease or a malignancy; beta-thalassemia; cystic fibrosis; earlyanemia of prematurity; anemia associated with chronic inflammatorydisease; spinal cord injury; acute blood loss; aging; and neoplasticdisease states accompanied by abnormal erythropoiesis.
 68. A methodaccording to claim 66, wherein each PEG has a molecular weight of about20,000 Daltons.
 69. A pharmaceutical composition comprising the compoundof claim 64 and a pharmaceutically acceptable carrier.
 70. Apharmaceutical composition according to claim 69, wherein each PEG has amolecular weight of about 20,000 Daltons.
 71. A compound that binds toand activates the erythropoietin receptor (EPO-R), which compoundcomprises a peptide dimer having the formula:

wherein (i) in each peptide monomer of the peptide dimer, each aminoacid is indicated by standard one letter abbreviation, AcG isN-acetylglycine, and 1-nal is 1-naphthylalanine; (ii) each peptidemonomer of the peptide dimer contains an intramolecular disulfide bondbetween the two cysteine (C) residues of each monomer (iii) PEGcomprises a linear unbranched polyethylene glycol (PEG) moiety having amolecular weight of about 20,000 to about 40,000 Daltons.
 72. A compoundthat binds to and activates the erythropoietin receptor (EPO-R), whichcompound comprises a peptide dimer having the formula:

wherein (i) in each peptide monomer of the peptide dimer, each aminoacid is indicated by standard one letter abbreviation, AcG isN-acetylglycine, and 1-nal is 1-naphthylalanine; (ii) each peptidemonomer of the peptide dimer contains an intramolecular disulfide bondbetween the two cysteine (C) residues of each monomer (iii) PEGcomprises two linear polyethylene glycol (PEG) moities having a combinedmolecular weight of about 10,000 to about 60,000 Daltons.